Novel insights into the mechanism of electron transfer in mitochondrial cytochrome c and its regulation by post-translational modifications

Instituto de Investigaciones Químicas cicCartuja, Universidad de Sevilla ─ CSIC Novel insights into the mechanism of electron transfer in mitochondrial cytochrome c and its regulation by post-translational modifications Memoria presentada por el Graduado D. Gonzalo Pérez Mejías para optar al título de Doctor por la Universidad de Sevilla con la acreditación de Mención Internacional. Sevilla, 2023 Directores Dr. D. Miguel Ángel de la Rosa Acosta Catedrático de la Universidad de Sevilla Dr. Dña. Irene Díaz Moreno Catedrática de la Universidad de Sevilla A mi familia 5 CONTENT Foreword ......................................................................................... 9 Abbreviations ................................................................................ 13 List of Publications and Merits of the PhD candidate ................. 19 Chapter 1: Introduction................................................................ 29 1.1 Mitochondrial electron transport chain .......................... 29 1.1.1 Electron transport chain complexes ............... 29 1.1.2 Electron transport chain organization ............. 33 1.1.3 Cytochrome c ................................................ 36 1.1.3.1 Role under homeostatic conditions . 36 1.1.3.2 Role under stress conditions ........... 39 1.2 Post-translational modifications .................................... 43 1.2.1 Acetylation .................................................... 45 1.2.2 Phosphorylation ............................................ 46 1.2.3 Role of post-translational modifications in cytochrome c functioning .......................... 47 Chapter 2: Objectives ................................................................... 53 Chapter 3: Results and Discussion ............................................... 57 3.1. Article I: Physical contact between cytochrome c 1 and cytochrome c increases the driving force for electron transfer .................................................................. 57 3.2. Article II: Exploring protein phosphorylation by combining computational approaches and biochemical methods .............................................. 61 3.3. Article III: Structural and functional insights into lysine acetylation of cytochrome c using mimetic point mutants .................................................................. 64 3.4. Article IV: Electric field-induced functional changes in electrode-immobilized mutant species of human cytochrome c.......................................................... 68 3.5. Article V: Phosphorylation disrupts long-distance electron transport in cytochrome c ......................... 71 Chapter 4: Conclusions ................................................................. 79 Chapter 5: Outlook and Future Perspectives ............................... 83 6 General References ....................................................................... 91 Appendix I: Scientific Articles .................................................... 129 Article I: Physical contact between cytochrome c 1 and cytochrome c increases the driving force for electron transfer ................................................................ 129 Article II: Structural and functional insights into lysine acetylation of cytochrome c using mimetic point mutants ................................................................ 157 Article III: Structural and functional insights into lysine acetylation of cytochrome c using mimetic point mutants ................................................................ 181 Article IV: Electric field-induced functional changes in electrode-immobilized mutant species of human cytochrome c........................................................ 219 Article V: Phosphorylation disrupts long-distance electron transport in cytochrome c .................................... 239 Appendix II: Review Articles ..................................................... 263 Review I: Novel insights into the mechanism of electron transfer in mitochondrial cytochrome c ................ 263 Review II: Post-translational modifications of cytochrome c in cell life and disease .......................................... 277 Review III: Wheel and deal in the mitochondrial inner membranes: the tale of cytochrome c and cardiolipin ............................................................................ 299 Review IV: Cytochrome c: Surfing off of the mitochondrial membrane on the tops of complexes III and IV ..... 321 7 FOREWORD 9 FOREWORD This document has been elaborated following the guidelines of the University of Seville on how to submit a PhD thesis as a compendium of scientific publications. It consists of the following sections: II. The list of Abbreviations. III. The list of Publications and Merits of the PhD candidate. IV. An Introduction justifying the thematic unit of the scientific publications presented. V. A section covering the Objectives of the PhD thesis. VI. A brief description of the Results and Discussion, including main achievements and outcomes. VII. A section covering the global Conclusions. VIII. A section covering the Outlook and Future Perspectives. IX. The list of References cited in previous sections. X. The Appendix I containing the five scientific articles on which the PhD thesis is based. All papers were published in scientific journals indexed in the Journal Citations Reports (JCR) database. XI. The Appendix II containing review articles co-authored by the PhD candidate and closely related to the PhD research work. 17 LIST OF PUBLICATIONS AND MERITS List of Publications and Merits of the PhD candidate 19 LIST OF PUBLICATIONS AND MERITS OF THE PHD CANDIDATE 1. Scientific publications  Gomila AMJ*, Pérez-Mejías G*, Nin-Hill A*, Guerra-Castellano A, Casas-Ferrer L, Ortiz-Tescari S, Díaz-Quintana A, Samitier J, Rovira C, De la Rosa MA, Díaz-Moreno I, Gorostiza P, Lagunas A, Giannotti M. 2022. “Phosphorylation disrupts long-distance electron transport in cytochrome c”. Nat. Commun., 13: 7100.  Marongiu M*, Pérez-Mejías G*, Orrù V, Steri M, Sidore C, Díaz- Quintana A, Mulas A, Busonero F, Maschio A, Walter K, Tardaguila M, Akbari P, Soranzo N, Fiorillo E, Gorospe M, Schlessinger D, Díaz- Moreno I, Cucca F, Zoledziewska M. 2022. “GWAS of genetic factors affecting white blood cell morphological parameters in Sardinians uncovers influence of chromosome 11 innate immunity gene cluster on eosinophil morphology”. Hum. Mol. Genet., 22: ddac238.  Olloqui-Sariego JL, Pérez-Mejías G, Márquez I, Guerra-Castellano A, Calvente JJ, De la Rosa MA, Andreu R, Díaz-Moreno I. 2022. “Electric field-induced functional changes in electrode-immobilized mutant species of human cytochrome c”. Biochim. Biophys. Acta - Bioenerg., 1863: 148570.  Pérez-Mejías G, Díaz-Quintana A, Guerra-Castellano A, Díaz-Moreno I, De la Rosa MA. 2022. “Novel insights into the mechanism of electron transfer in mitochondrial cytochrome c”. Coord. Chem. Rev., 450: 214233.  Márquez I, Pérez-Mejías G, Guerra-Castellano A, Olloqui-Sariego JL, Andreu R, Calvente JJ, De la Rosa MA, Díaz-Moreno I. 2021. “Structural and functional insights into lysine acetylation of cytochrome c using mimetic point mutants”. FEBS Open Bio, 11: 3304– 3323. Gonzalo Pérez Mejias 20  González-Arzola K, Guerra-Castellano A, Rivero-Rodríguez F, Casado-Combreras MÁ, Pérez-Mejías G, Díaz-Quintana A, Díaz- Moreno I, De la Rosa MA. 2021. “Mitochondrial cytochrome c shot towards histone chaperone condensates in the nucleus”. FEBS Open Bio, 11: 2418–2440.  Elena-Real CA, González-Arzola K, Pérez-Mejías G, Díaz-Quintana A, Velázquez-Campoy A, Desvoyes B, Gutiérrez C, De la Rosa MA, Díaz-Moreno I. 2021. “Proposed mechanism for regulation of H 2 O 2 - induced programmed cell death in plants by binding of cytochrome c to 14-3-3 proteins”. Plant J., 106: 74–85.  Guerra-Castellano A, Márquez I, Pérez-Mejías G, Díaz-Quintana A, De la Rosa MA, Díaz-Moreno I. 2020. “Post-translational modifications of cytochrome c in cell life and disease”. Int. J. Mol. Sci., 21: 8483.  Pérez-Mejías G, Velázquez-Cruz A, Guerra-Castellano A, Baños- Jaime B, Díaz-Quintana A, González-Arzola K, De la Rosa MA, Díaz- Moreno I. 2020. “Exploring protein phosphorylation by combining computational approaches and biochemical methods”. Comput. Struct. Biotechnol. J., 18: 1852–1863.  Pérez-Mejías G, Olloqui-Sariego JL, Guerra-Castellano A, Díaz- Quintana A, Calvente JJ, Andreu R, De la Rosa MA, Díaz-Moreno I. 2020. “Physical contact between cytochrome c 1 and cytochrome c increases the driving force for electron transfer”. Biochim. Biophys. Acta - Bioenerg., 1861: 148277.  Díaz-Quintana A, Pérez-Mejías G, Guerra-Castellano A, De la Rosa MA, Díaz-Moreno I. 2020. “Wheel and deal in the mitochondrial inner membranes: the tale of cytochrome c and cardiolipin”. Oxid. Med. Cell. Longev., 2020: 6813405. List of Publications and Merits of the PhD candidate 21  González-Arzola K, Velázquez-Cruz A, Guerra-Castellano A, Casado- Combreras MÁ, Pérez-Mejías G, Díaz-Quintana A, Díaz-Moreno I, De la Rosa MÁ. 2019. “New moonlighting functions of mitochondrial cytochrome c in the cytoplasm and nucleus”. FEBS Lett., 593: 3101– 3119.  Pérez-Mejías G, Guerra-Castellano A, Díaz-Quintana A, De la Rosa MA, Díaz-Moreno I. 2019. “Cytochrome c: Surfing off of the mitochondrial membrane on the tops of complexes III and IV”. Comput. Struct. Biotechnol. J., 17: 654–660.  Guerra-Castellano A, Díaz-Quintana A, Pérez-Mejías G, Elena-Real CA, González-Arzola K, García-Mauriño SM, De la Rosa MA, Díaz- Moreno I. 2018. “Oxidative stress is tightly regulated by cytochrome c phosphorylation and respirasome factors in mitochondria”. Proc. Natl. Acad. Sci. U.S.A., 115: 7955–7960. 2. Congress communications Oral communications  Role of physical contacts and long-range electron tunneling in the mechanism of mitochondrial cytochrome c electron transfer. Pérez-Mejías G, Guerra-Castellano A, Olloqui-Sariego JL, Giannotti M, Lagunas A, Gomila A, Nin-Hill A, Calvente JJ, Andreu R, Gorostiza P, De la Rosa MA, Díaz-Moreno I. XVIII Simposio de Jóvenes Investigadores Químicos RSEQ. November 21 st -24 th 2022, Sevilla (Spain). Gonzalo Pérez Mejias 22  Novel insights into the mechanism of electron transfer in mitochondrial cytochrome c. Pérez-Mejías G, Díaz-Quintana A, Guerra-Castellano A, Díaz-Moreno I, De la Rosa MA. IUBMB Focused Meeting / FEBS Workshop Crosstalk between Nucleus and Mitochondria in Human Disease. March 22 nd -25 th 2022, Sevilla (Spain).  Molecular basis for acute hypoxia-related diseases: Understanding the cross-talk between cytochrome c and hypoxia-induced protein HIGD1A in the mitochondria. Pérez-Mejías G, Guerra-Castellano G, Díaz-Quintana A, González- Arzola K, De la Rosa MA, Díaz-Moreno I. 44 th FEBS Congress. July 6 th -11 th 2019, Krakow (Poland).  Molecular basis for acute hypoxia-related diseases: Understanding the cross-talk between cytochrome c and hypoxia-induced protein HIGD1A in the mitochondria. Pérez-Mejías G, Guerra-Castellano G, Díaz-Quintana A, González- Arzola K, De la Rosa MA, Díaz-Moreno I. 19 th FEBS Young Scientist’ Forum. July 3 rd -6 th 2019, Krakow (Poland). Poster presentations  Impact of post-translational modifications and disease-related mutations on the structural dynamic properties of cytochrome c. Pérez-Mejías G, Márquez I, Lasorsa A, Morse PT, Turner AA, De la Rosa MA, Hüttemann M, van der Wel PCA, Díaz-Moreno I. European Conference on Magnetic Resonance – EUROMAR 2022. July 10 th -14 th 2022, Utrecht (Netherlands). List of Publications and Merits of the PhD candidate 23  Physical contact between cytochrome c 1 and cytochrome c increases the driving force for electron transfer. Pérez-Mejías G, Olloqui-Sariego JL, Guerra-Castellano A, Díaz- Quintana A, Calvente JJ, Andreu R, De la Rosa MA, Díaz-Moreno I. 43 rd SEBBM Congress. July 19 th -22 nd 2021, Barcelona (Spain).  Exploring protein phosphorylation by combining computational approaches and biochemical methods. Pérez-Mejías G, Velázquez-Cruz A, Guerra-Castellano A, Baños- Jaime B, Díaz-Quintana A, González-Arzola K, De la Rosa MA, Díaz- Moreno I. 7 th International Iberian Biophysics Congress. June 14 th -16 th 2021, Coimbra (Portugal).  Role of HIGD1A and cytochrome c protein in mitochondrial respiratory chain supercomplexes. Pérez-Mejías G, Guerra-Castellano A, Díaz-Quinana A, Mallou- Roncero E, Ponce-España E, De la Rosa MA, Díaz-Moreno I. 16 th Congress of the Spanish Biophysical Society. June 6 th -8 th 2017, Sevilla (Spain). 3. Congresses and courses organization  IUBMB Focused Meeting / FEBS Workshop Crosstalk between Nucleus and Mitochondria in Human Disease. March 22 nd -25 th 2022, Sevilla (Spain). Role: Organizing committee. Gonzalo Pérez Mejias 24  FEBS Practical Course on Integrated Approaches to Biomolecular Interactions. May 7 th -11 th 2018, Sevilla (Spain). Role: Organizing committee and tutor during hands-on training sessions.  16 th Congress of the Spanish Biophysical Society. June 6 th -8 th 2017, Sevilla (Spain). Role: Organizing committee. 4. International research training  Zernike Institute for Advanced Materials, solid state NMR group, University of Groningen, the Netherlands. 23 weeks, 2020-2021. Supervisor: Prof. Patrick C. A. van der Wel. Project: Impact of post-translational modifications on the interaction between human cytochrome c and cardiolipin.  Institute of Structural Biology, Cell-free protein expression unit, Grenoble, France. 1 week, 2018. Supervisor: Lionel Imbert. Project: PID2921 - Respiratory supercomplexes: the interaction between hypoxia-inducible proteins and cytochrome c in humans. 5. Bursaries and awards  PhD fellowship from the Spanish Ministry of Education, Culture and Sports. Reference number FPU017/04604, 2018-2023.  Roche award to attend Roche Continents, 2022.  Travel grant to attend the European Conference on Magnetic Resonance – EUROMAR, 2022. List of Publications and Merits of the PhD candidate 25  Short-Term Fellowship of the Federation of European Biochemical Societies (FEBS) to visit the Zernike Institute for Advanced Materials (Groningen, the Netherlands), 2021.  Award “Paper of the Month” of the Spanish Biophysics Society (SBE) for scientific article published in Computational and Structural Biotechnology Journal, 2020.  Short-Term Fellowship of the European Molecular Biology Organization (EMBO) to visit the Zernike Institute for Advanced Materials (Groningen, the Netherlands), 2020.  Young Scientists’ Forum award to attend the 19 th Young Scientists’ Forum and 44 th FEBS Congress, 2019.  Research Initiation Fellowship from the Spanish Ministry of Education, Culture and Sports, 2016. Gonzalo Pérez Mejias 32 transferred to the heme group of Cyt c 1 through a conformational change of the Rieske protein, which contains a [Fe 2 -S 2 ] cluster, and it is subsequently donated to Cyt c (Darrouzet et al., 2000; 2002; Kokhan et al., 2010). The second electron travels across the Cyt b subunit to the Q i site to reduce a bound oxidized Q molecule, yielding a semiquinone intermediate, which is fully reduced to hydroquinone in a second cycle (Figure 3). Figure 3. Complex III and IV cofactors and function. Cofactors and metal centers involved in ET are highlighted within the structure of each complex, and their chemical formulas are shown in colored boxes. Structure of CIII (PDB code 5XTE), Cyt c (PDB ID 2N9I) and CIV (PDB code 5XTH). At the last step of the ETC, reduced Cyt c molecules are oxidized by CIV, and the electrons are donated to molecular oxygen, generating water (Ruitenberg et al., 2002; Belevich et al., 2006). Such a process involves four metallic cofactors (a binuclear copper cofactor Cu A , a heme a, a high-spin heme a 3 , and a mononuclear copper cofactor Cu B ) and is coupled with the translocation of four protons per O 2 molecules across the IMM (Tsukihara et al., 1996; Ruitenberg et al., 2002; Belevich et al., 2006) (Figure 3). Regarding the structure of CIV, recent findings demonstrate that CIV can be found in three different states (dimer, independent monomer, and monomer within supercomplexes structures) and its activity might be regulated by the equilibrium relationship between them (Shinzawa-Itoh et al., 2019; Sedlák et Introduction 33 al., 2020). Each monomer is composed of 10-14 subunits in eukaryotic cells, including several growth condition- and tissue-specific isoforms. Additional factors, including COX18, are necessary for the CIV core assembly (Bourens and Barrientos, 2017). Recent research has revealed that NDUFA4, which was long thought to be a CI subunit and latter a CIV regulatory factor, is a mammalian CIV subunit (the so-called 14 th subunit) (Balsa et al., 2012; Kadenbach, 2017; Pitceathly and Taanman, 2018; Zong et al., 2018). 1.1.2 Electron transport chain organization The dynamics and organization of the intermembrane ETC complexes within the mitochondria have been a matter of debate over the last decades. One of the first proposed model was the “solid model”, in which all the respiratory mitochondrial membrane complexes are assembled into a high-order structure, named oxysome, with the electron carriers bound to the complexes (Chance and Williams, 1955) (Figure 4a). In contrast, the “fluid model” states that the ETC complexes diffuse freely and independently in the IMM, and are connected by freely diffusing electron carriers (Hackenbrock et al., 1976; 1986) (Figure 4b). At the beginning of this century, the development of blue native acrylamide gel electrophoresis (BN-PAGE) technique allowed the isolation of supramolecular functional mitochondrial enzyme units, namely supercomplexes (SCs) (Schägger, 2000). Based on earlier observations by Ferguson-Miller and coworkers (Ferguson-Miller et al., 1976), the “plasticity model” was proposed to harmonize both models (solid and fluid) (Acín-Pérez and Enriquez, 2014). This model considers a balanced distribution between SCs and free complexes to fit the demanding cellular needs. Indeed, SCs have been found in various organisms, ranging from bacteria and archaea, to plants and mammals (Schägger, 2000; 2002; Eubel et al., 2004; Krause et al., 2004; Stroh et al., 2004; Schäfer et al., 2006; Maas et al., 2009; Suthammarak et al., 2013; Melo and Teixeira, 2016; Davies et al., 2018; Wiseman et al., 2018). In the Gonzalo Pérez Mejias 34 frame of the “plasticity model”, several studies have demonstrated the dynamic behavior and heterogeneity of the stoichiometries and degree of association between each respiratory complex within the SCs, as they can be modulated by the cell type, cellular condition, and tissue-specific isoforms (Fukuda et al., 2007; Ikeda et al., 2013; Genova and Lenaz, 2014; Sun et al., 2016; Greggio et al., 2017). Figure 4. Organization models for electron transport chain. Schematic representation of the solid (A) and fluid (B) models to explain the organization of the ETC. Recently, an increasing number of eukaryotic CIII/CIV-containing SCs structures have been solved with a near-atomic resolution, owing to the development of cryogenic electron microscopy (cryo-EM) (Wu et al., 2016; Letts et al., 2016; Sousa et al., 2016; Guo et al., 2017; Hartley et al., 2019; Rathore et al., 2019; Berndtsson et al., 2020; Maldonado et al., 2021; Moe et al., 2021; Vercellino and Sazanov, 2021; Jeon et al., 2022). One of the most common and studied SCs in mammals is the so-called respirasome, which is assembled by CI, CIII, and CIV and is capable of performing in vitro NADH:O 2 oxidoreduction (Wu et al., 2020). Introduction 35 The exact mechanism of assembly of SCs is not yet fully understood and several models have been proposed. On one hand, it was proposed that intermediates of individual complexes act as a scaffold for the assembly of the SCs (Moreno-Lastres et al., 2012). On the contrary, other model proposed that SCs are originated from preassembled individual complexes (Acín-Pérez et al., 2008). Based on recent observations, it has been proposed that SC biogenesis takes place through a cooperative assembly mechanism, in which the so-called supercomplex assembly factors (SCAFs) act as regulatory checkpoints at specific assembly steps (Protasoni et al., 2020; Fernández-Vizarra and Ugalde, 2022). Among the possible SCAFs, several proteins have been proposed to have a role in the assembly and stability of SCs in mammals, i.e., cytochrome c oxidase subunit 7A-like 2 (COX7AL2, a.k.a. SCAF1) and hypoxia-induced gene 1 domain (HIGD) family member 1A and 2A (HIGD1A and HIGD2A) (Chen et al., 2012; Pérez-Pérez et al., 2016; Lobo-Jarne et al., 2018; Salazar et al., 2019; García‐Poyatos et al., 2020; Lobo‐Jarne et al., 2020; Timón-Gómez et al., 2020). The lipid composition of the mitochondrial membrane has indeed an important role in the organization and formation of SCs, e.g., the lack of cardiolipin impacts the stability of the respiratory complexes and SCs formation (Zhang et al., 2002; Pfeiffer et al., 2003; McKenzie et al., 2006; Mileykovskaya and Dowhan, 2014). Even though the existence of SCs has been widely supported by experimental data, several roles have been proposed and no consensus has been reached on their physiological implications (Barrientos and Ugalde, 2013; Letts and Sazanov, 2017; Milenkovic et al., 2017; Lobo-Jarne and Ugalde, 2018; den Brave and Becker, 2020; Brzezinski et al., 2021; Vercellino and Sazanov, 2022). Some of the roles that have been proposed include increasing the stability of individual complexes, damping the production of reactive oxygen species (ROS), avoiding aggregation due to overcrowding, or enhancing the ET activity of individual complexes. Gonzalo Pérez Mejias 36 1.1.3 Cytochrome c 1.1.3.1 Role under homeostatic conditions Cyt c is a small soluble positively-charged monoheme protein (ca. 12 kDa, 104 amino acids, and an isoelectric point of 9.5 in humans) found in the IMS under homeostatic conditions. Cyt c three-dimensional (3D) structure consists of five cooperative folding-unfolding units (known as foldons), composed of four α-helices and two extended loops (Maity et al., 2004) (Figure 5a). Figure 5. Structure of human cytochrome c. A) Ribbon representation of Cyt c human structure (PDB code 2N9I). The different foldon units (left panel) are colored as follow: foldon I in blue, foldon II in green, foldon III in yellow, foldon IV in red, and foldon V in purple. Right panel: heme group, axial ligands, α-helixes, and N- and C-ends are labeled. B) Coulombic surface potential (left) and hydrophobicity (right) of human Cyt c, highlighting the positive ring around the hydrophobic patch that surrounds the heme cleft (green). Introduction 37 Cyt c possesses a c-type heme group that is covalently attached through disulfide bonds to the polypeptide chain by the consensus sequence Cys-X-X- Cys-His. The polypeptide chain is wrapped around the heme group establishing aromatic and hydrophobic interactions. Therefore, the heme group is only partially exposed to the solvent, allowing an efficient ET with its redox partners. Under homeostatic and physiological conditions, the iron atom of the heme group, which can be found in two different redox states (ferrous state Fe 2+ or ferric state Fe 3+ ), is hexacoordinated, being the axial ligands the His18 and Met80 residues (Figure 5a). The main function of Cyt c under homeostatic conditions is to act as a mobile electron carrier between CIII and CIV. Cyt c binds to its redox partners via electrostatic interactions, involving the lysine-rich positively-charged area surrounding the Cyt c heme cleft and the complementary negatively-charged patches on CIII and CIV (Speck et al., 1979; Capaldi et al., 1982; Lange and Hunte, 2002; Nyola and Hunte, 2008; Shimada et al., 2017) (Figure 5b). Originally, it was proposed that the diffusion of Cyt c between CIII and CIV occurs predominantly via a general pool of Cyt c molecules in the IMS (Gupte and Hackenbrock, 1988). In this context, Cyt c diffuses in all dimensions with no restrictions between its redox partners (3D diffusion) (Figure 6a). However, several studies have challenged this model, supporting the hypothesis that Cyt c can diffuse laterally along the respiratory complexes or the IMM (bidimensional 2D sliding) (Figure 6b). Nuclear magnetic resonance (NMR) studies combined with isothermal titration calorimetric (ITC) approaches described the existence of additional low-affinity Cyt c-binding sites on the surface of plant CIII and mammalian CIV (Moreno-Beltrán et al., 2014; 2015), which were in agreement with old kinetic studies (Ferguson- Miller et al., 1976). These recently reported additional binding sites are nonproductive for ET (also known as distal binding sites) and have been proposed to act as landing or taking-off platforms for Cyt c molecules laterally diffusing between CIII and CIV (Pérez-Mejías et al., 2019). Indeed, recent cryo-EM data Gonzalo Pérez Mejias 38 evidenced an elongated Cyt c density along the negatively-charged path that connects Cyt c bindings sites in CIII and CIV, with equal probability of finding Cyt c bound to CIII, bound to CIV, or occupying the area between both binding sites (Moe et al., 2021). Moreover, theoretical analysis of electron flow between CIII and CIV mediated by Cyt c concluded that the shortening of the average distance between CIII and CIV by SC formation might solve the diffusion-limitation problem, as the time to find the destination can be exceptionally large in bidimensional systems (Stuchebrukhov et al., 2020). Therefore, the presence of additional binding sites and the electrostaticallynegative surface platform between CIII and CIV might be a way of raising the probability of Cyt c 2D sliding onto CIII and CIV, facilitating the ET. Figure 6. Cytochrome c diffusion between its redox partners. Cyt c can either diffuse in all dimensions with no restrictions (A) or diffuse laterally along the respiratory complexes or the IMM (B). Besides its main function as a mobile electron carrier between CIII and CIV, Cyt c acts as ROS scavenger (Korshunov et al., 1999; Wang et al., 2005; Pasdois et al., 2011) and takes part in the redox-coupled import of cysteine-rich proteins via Erv1-Mia40 pathway (Allen et al., 2005; Dabir et al., 2007; Díaz- Moreno et al., 2011a; Volkov and van Nuland, 2012). Introduction 39 In this context, Cyt c functions as an electron carrier between Erv1 and Cyt c peroxidase, coupling Cyt c peroxidase reduction and Erv1 oxidation cycles. Cyt c-Cyt c peroxidase complex features a highly dynamic behavior with two nearly equally populated states—the ET-compatible stereo-specific state and the transient intermediate encounter state (Bashir et al., 2010; Son et al., 2020). A recent NMR study showed that binding and ET between Erv1 and Cyt c is a collision-type rather than an electrostatically-driven interaction (Peker et al., 2021). Cyt c reductase activity of Erv1 is regulated by its degree of association with the mitochondrial membrane, being higher when Erv1 is soluble in the IMS than when associated to the mitochondrial membrane (Tang et al., 2020). These data suggest that the Cyt c population that would be involved in the interaction and ET with these other redox partners would be the soluble population of Cyt c in the IMS bulk, rather than the population associated with the SCs or the IMM. 1.1.3.2 Role under stress conditions Besides its well-established role in cellular respiration and mitochondrial metabolism, Cyt c has an important role in cell fate and death (González-Arzola et al., 2019). Under homeostatic conditions, cardiolipin (CL) shows an asymmetrical distribution in the IMM, being more abundant in the internal leaflet facing the mitochondrial matrix (Krebs et al., 1979). However, under stress conditions, CL is translocated from the IMM to the outer mitochondrial membrane (OMM), becoming a signaling hub for programmed cell death (apoptosis) and mitophagy (Garcia Fernandez et al., 2002; Chu et al., 2013). Rearrangement of CL increases the availability for Cyt c binding, which is capable of oxidizing the acyl chains of CL due to its peroxidase activity (Belikova et al., 2006). Cyt c binding to CL has been extensively studied over the last fifty years and several binding sites have been described (Figure 7a) (Díaz-Quintana et al., 2020). Positively charged and hydrophobic Cyt c Gonzalo Pérez Mejias 40 residues are common features of these binding sites. The formation of the complex is initiated by electrostatic interactions between positive residues of Cyt c and the negatively charged phosphate groups of CL (Figure 7b). After complex formation, hydrophobic Cyt c residues are key to establishing tight interaction with CL acyl chains (Figure 7c). Figure 7. Cardiolipin binding sites in cytochrome c. A) Ribbon representation of Cyt c human structure (PDB code 2N9I). The different CL-binding sites are colored as follow: A-site or distal productive in orange, N-site or proximal unproductive in blue, C-site in magenta, and L-site or proximal productive in green. Representation of positively charged Cyt c residues (B) involved in the formation of the Cyt c-CL complex, and hydrophobic Cyt c residues (C), which ensure tight interaction between Cyt c and CL acyl chains. Several authors have proposed that such interaction destabilizes or unfolds the protein (Belikova et al., 2006; Sinibaldi et al., 2010; Muenzner et al., 2013). However, it is still unclear how lipid binding influences Cyt c structure. The reason is that recent reports have shown no sign of a molten Introduction 41 globule or unfolding of Cyt c upon binding to cardiolipin-containing vesicles (Mandal et al., 2015; Mohammadyani et al., 2018; Li et al., 2019). An increase in the levels of oxidized CL favors the release of Cyt c from the mitochondria into the cytoplasm (Shidoji et al., 1999; Petrosillo et al., 2003; Kagan et al., 2005). Cyt c release involves the movement through the OMM, which is impermeable for most proteins. Several mechanisms have been proposed to explain how Cyt c is released into the cytosol (Figure 8) (Gogvadze et al., 2006; Ow et al., 2008). The first mechanism relies on the opening of non-specific pores in the IMM that causes osmotic swelling of the mitochondrial matrix, rupture of the OMM and release of several intermembrane proteins, including Cyt c (Yang and Cortopassi, 1998). Similarly, B-cell lymphoma protein-2 (Bcl-2) protein family members can form pores and channels in the OMM. After a conformational change of Bcl-2-associated X protein (BAX) and Bcl-2 homologous antagonist killer (BAK) triggered by apoptotic stimuli, these proteins are inserted into the OMM, forming ring-like oligomeric structures that allow the release of Cyt c and other intermembrane proteins (Große et al., 2016; Salvador‐Gallego et al., 2016). Finally, a third proposed mechanism involves oligomerization of the voltage-dependent anion channel protein (VDAC) (Zalk et al., 2005; Keinan et al., 2013; Trindade et al., 2016). Cyt c translocation to the cytoplasm and binding to apoptosis-inducing factor 1 (Apaf-1) are considered a critical point of no return in the execution of apoptosis. The hetero-oligomeric complex formed by Cyt c and Apaf-1—the so-called apoptosome—cleaves procaspase-9 to activate the caspase cascade through procaspase-3 cleavage, triggering apoptosis (Figure 8) (Cai et al., 1998). Furthermore, cytosolic Cyt c can interact with a wide variety of proteins to block pro-survival pathways and induce apoptotic pathways (Boehning et al., 2003; Martínez-Fábregas et al., 2013; 2014; Elena-Real et al., 2018; 2021). Gonzalo Pérez Mejias 48 Figure 9. Post-translational modifications of cytochrome c. Ribbon representation of human Cyt c structure (PDB code 2N9I) with side chains of amino acids that undergo nitration (A), acetylation (B) and phosphorylation (C) labeled in turquoise, orange, and purple, respectively. Thr58 is replaced by isoleucine in human Cyt c. As described before, Cyt c is a highly positive protein (eighteen lysine residues) that binds its redox partners via electrostatic interactions, involving the lysine-rich positively-charged area surrounding the heme cleft. Several studies have explored the in vitro properties of chemically acetylated Cyt c, showing a decrease in Cyt c-mediated ET (Minakami et al., 1958; Wada and Okunuki, 1968; Azzi et al., 1975). Indeed, acetylation of more than six lysine residues leads to a complete loss of Cyt c ability to interact with and transfer an electron to CIV (Wada and Okunuki, 1968). However, only two residues of mammalian Cyt c (Lys8 and Lys53) have been reported to be acetylated in vivo (Figure 9b). Lys8 was detected in a high-throughput proteomics study in mouse liver mitochondria under fasting conditions (Kim et al., 2006). On the Introduction 49 other hand, Lys53 acetylation was identified in prostate tumor xenografts in mice (Bazylianska et al., 2021) and the functional implications were analyzed with acetyl mimetic rodent Cyt c. Downregulation of mitochondrial ET by Lys53 acetylation has been linked to the “Warburg effect” (cell respiration inhibition) experienced by cancer cells, where metabolic reprogramming favors cell proliferation (Hanahan and Weinberg, 2011). Tissue-specific phosphorylation of six different Cyt c residues has been detected in vivo on mammalian Cyt c, i.e., Thr28, Ser47, Tyr48, Thr49, Thr58, and Tyr97 (Figure 9c). Under basal conditions, Thr28 was detected in bovine kidney (Mahapatra et al., 2017), Ser47 in porcine brain (Kalpage et al., 2019b), Tyr48 in bovine liver (Yu et al., 2008), Thr58 in murine kidney (Wan et al., 2019), and Tyr97 in bovine heart (Lee et al., 2006). Tyr97 phosphorylation was also detected in porcine brain after neuroprotective treatment with insulin after ischemia/reperfusion injury (Sanderson et al., 2013). Thr28 and Ser47 were also found to be phosphorylated in human skeletal muscle under basal conditions and after exercise by high-throughput phosphoproteomics study (Zhao et al., 2011; Hoffman et al., 2015). The level of Thr49 phosphorylation has been recently found to be increased upon aging as detected by a proteomics study in myocardial tissue from young and old mice (Li et al., 2022). Among the different phosphorylations that Cyt c can suffer, the corresponding kinases have been only identified for Thr28 and Ser47 phosphorylation. Adenosine 5’-monophosphate (AMP)-activated kinase is responsible for Thr28 phosphorylation (Mahapatra, et al. 2017), while Akt kinases were identified as potential kinases mediating Ser47 phosphorylation (Kalpage et al., 2020). The effects of PTMs of Cyt c have been usually studied with phosphomimetic proteins, as obtaining phosphoproteins from natural sources has limitations in terms of sample quantity and stability. The effect of Ser/Thr phosphorylation has been analyzed with traditional phosphomimetic amino acid substitution (Asp/Glu) (Guerra-Castellano et al., 2016; Kalpage et al., 2019b; Wan et al., 2019; Li et al., 2022), whereas Tyr phosphorylation has Gonzalo Pérez Mejias 50 been assessed by non-canonical amino acid substitution (Guerra-Castellano et al., 2015; 2018; Moreno-Beltrán et al., 2017). Functional studies have shown that phosphorylation of Cyt c leads to partial inhibition of ETC, reduced ROS production, and mitochondrial membrane potential. Moreover, all the phosphorylation sites of Cyt c—except Thr28—impair its ability for caspase-3 activation via the apoptosome. The functional consequences of PTMs in the Cyt c nuclear roles recently described are still unexplored. However, based on the nature of the interactions of Cyt c with its nuclear partners, which involves acidic disordered stretches, a potential role for acetylation and phosphorylation of Cyt c as inhibitory modifications can be proposed. Recent studies have demonstrated that acidic domains of proteins, like SET/TAF-Iβ, may act as “readers” for unacetylated p53 (Wang et al., 2016; 2017). According to these authors, "readers" of unacetylated p53 C-terminal domain could potentially recognize other lysine-rich domains as well, being Cyt c an excellent candidate due to its basic and lysine-rich nature. 51 CHAPTER 2 OBJECTIVES “Sometimes science is more art than science, Morty. A lot of people don’t get that.” — Rick Sanchez, Rick and Morty Objectives 53 CHAPTER 2: OBJECTIVES The main objective of this PhD thesis is to study the electron transfer in the human cytochrome c 1 -cytochrome c complex and how post-translational modifications affect its function in the mitochondria. The specific objectives of this research are as follow: 1. To determine the biophysical and structural properties of the transient complex between human cytochrome c and the soluble domain of human cytochrome c 1 , and to understand the underlying mechanisms of electron transfer between cytochrome c 1 and cytochrome c. 2. To develop a workflow to assist researchers in choosing the most suitable alternative for studying the functional effects of protein phosphorylation by combining computational and biochemical approaches. 3. To analyze the changes in the biophysical, structural, and functional properties of cytochrome c induced by acetylation at positions 8 and 53, using Lys-to-Gln substitution to mimic lysine acetylation. 4. To ascertain the thermodynamics and electron exchange kinetics of surface-immobilized cytochrome c variants at varying local electric field. In particular, to evaluate the effects of phosphorylation at positions 48 and 97, as well as the naturally occurring Tyr-to-His mutation at position 48, which is related with thrombocytopenia. 5. To investigate the molecular mechanism of electron transfer regulation by Tyr48 phosphorylation of cytochrome c in complex with cytochrome c 1 . 55 CHAPTER 3 RESULTS AND DISCUSSION “The purpose of a storyteller is not to tell you how to think, but to give you questions to think upon.” — Brandon Sanderson, The Way of Kings Results and Discussion 57 CHAPTER 3: RESULTS AND DISCUSSION In this section, a quick overview of the Results and Discussion of published work is presented. Please, refer to the journal publications in Appendix I and Appendix II for further details. 3.1. Article I: Physical contact between cytochrome c 1 and cytochrome c increases the driving force for electron transfer CIII is responsible for the ET from the Q pool embedded in the IMM to soluble Cyt c and extensive work has been done to understand its physiological functioning. The redox reactions at the reducing side of the complex and the ET to Cyt c have been characterized in bacterial, yeast, and plant systems (Smith et al., 1981; Engstrom et al., 2003; Janzon et al., 2008; Nyola and Hunte, 2008; Sarewicz et al., 2008; Moreno-Beltrán et al., 2014; 2015). However, the structural changes and thermodynamic parameters driving ET in human mitochondria from the Rieske protein to Cyt c via Cyt c 1 are not fully understood yet. In Pérez-Mejías et al. (2020a), we characterized the physicochemical and redox properties of the water-soluble domain of human Cyt c 1 and its molecular recognition by human Cyt c. In order to recombinantly produce and purify the human version of Cyt c 1 , we cloned the N-terminal water-soluble domain of Cyt c 1 , removing the C-terminal hydrophobic helix following previous studies with soluble domains of Cyt c 1 from other sources (Li et al., 1981; Konishi et al., 1991; Meyer et al., 2011; Moreno-Beltrán et al., 2014). The recombinant soluble Cyt c 1 domain exhibited proper folding and heme coordination as inferred from circular dichroism (CD), NMR, and absorption spectra. We analyzed Cyt c 1 redox properties by performing cyclic voltammetric experiments onto polycrystalline gold electrodes modified with thiol self-assembled monolayers (SAMs) bearing different terminal groups, which allow for different modes of protein binding. Specifically, we used three Gonzalo Pérez Mejias 64 3.3. Article III: Structural and functional insights into lysine acetylation of cytochrome c using mimetic point mutants In Márquez et al. (2021), we analyzed the physicochemical, redox, and functional properties of human acetyl mimetic Cyt c species in positions 8 and 53, as there was no information regarding their effect on the human variants (Kim et al., 2006; Bazylianska et al., 2021). We relied on Lys-to-Gln substitution to mimic lysine acetylation because of the similar surface electrostatic potentials of acetyl lysine variants and glutamine mutants (Figure 15). Figure 15. Electrostatic surface potential of cytochrome c variants. Electrostatic potentials were calculated at 150 mM ionic strength for WT and residue 8 and 53 mutant species of Cyt c. Residue 8 (upper) and 53 (lower) are marked with green circles. Physicochemical characterization (CD and visible absorption spectroscopy) of the herein studied variants revealed the typical hallmarks of ctype cytochromes in all mutants, confirming that Lys8 and Lys53 mutations Results and Discussion 65 have a negligible effect on the heme environment. Trajectory analysis from MD simulations also supported the preservation of the heme moiety as the Met80- Fe bond distance and heme group solvent-accessible surface are conserved. Moreover, the E 1/2 values for the three Cyt c species (WT and acetyl mimetics) were not significantly different, thus suggesting that the acetyl mimetic mutations do not substantially modify the heme moiety. CSP analysis from the recorded NMR spectra revealed that the Lys-to- Ala mutation—which was used as a control mutation—resulted in substantial perturbation as compared to the Lys-to-Gln one; not only in terms of the magnitude of the CSP but also in terms of the number of affected residues (Figure 16). Alanine differs from lysine not only in the loss of charge, but also in the side-chain length. Moreover, alanine has the highest helix propensity while lysine and glutamine present similar lower values (Pace and Scholtz, 1998; Fujiwara et al., 2012). Therefore, Lys-to-Ala substitution could a induce higher perturbation than expected for a control mutation in the neighborhood of residues close to the new alanine residue. Regarding the surface map comparison of NMR CSPs of acetyl mimetic Cyt c mutants, the acetyl mimetic variant at position 8 (K8Q) showed a smaller number of affected residues with respect to the acetyl mimetic variant at position 53 (K53Q). Only residues located in the same helix I, as residue 8, experienced significant CSPs (K8Q variant). On the contrary, loss of charge at position 53 caused by glutamine substitution alters the chemical environment of nearby residues located in helix II as residue 53 and those located in the nested Ω-loop, which is close in space (Figure 16). Gonzalo Pérez Mejias 66 Figure 16. NMR chemical-shift perturbation of Lys8 and Lys53 cytochrome c variants. Bar plot of NMR chemical-shift perturbation (Δδ avg ) calculated from 2D 1 H- 15 N HSQC spectra of Lys8 (K8A and K8Q, left) and Lys53 (K53A and K53Q, right) variants with respect to 2D 1 H- 15 N HSQC WT spectrum. The different analyzed Cyt c species displayed similar electron exchange rates (1100-1600 s -1 ) between the protein and the polycrystalline gold electrode modified with negatively charged mixed SAMS. Interestingly, acetyl mimetic variants drove a higher activation enthalpy, whose effect is partially offset by an increase in the pre-exponential factor. Such higher activation enthalpy and pre-exponential factor values can be ascribed to greater participation of the solvent in the activation process, due to increased dynamics of the immobilized protein, and/or to a more flexible molecular envelope generated by a looser protein electrostatic absorption. Indeed, analysis of MD trajectories showed that root mean square fluctuations (RMSF) values of several stretches in the two acetyl mimetics are higher than those of WT Cyt c, which could explain the increase in the pre-exponential factor of immobilized acetyl mimetic mutants. Results and Discussion 67 Moreover, we analyzed the impact of these two acetyl mimetic variants on the mitochondrial Cyt c oxidase activity. Our data revealed a decrease in the activity for both acetyl mimetic species compared to the WT protein (Figure 17). These results were consistent with the in vitro ET rate of mouse K53Q to CIV recorded by Bazylianska and colleagues (2021), and with the presence of Lys8 at the interface of the Cyt c-CIV complex previously reported (Rieder and Bosshard, 1980; Sato et al., 2016; Shimada et al., 2017). Even though the effect on the activity is similar for both acetyl mimetic species, the underlying mechanism might be different. While Lys8 is involved in the ET-compatible proximal Cyt c binding site of CIV, Lys53 is located at the interface of the distal Cyt c CIV binding site. Such findings suggest that Lys8 acetylation could modulate the ET to CIV by affecting the recognition mode of Cyt c to the primary ET active binding site, whereas acetylation of Lys53 could slow down the turnover of Cyt c molecules as a result of changes in the distal binding site (Figure 17). Figure 17. Modulation of cytochrome c oxidase activity. In vitro Cyt c oxidase activity from mouse embryonic fibroblast-isolated mitochondrial extracts mediated by Cyt c variants (left). Representative structure of Cyt c at the proximal and distal binding sites of Cyt c oxidase complex (right). Lys8 is colored in blue and Lys53 is colored in green. These results lay the groundwork for more detailed studies on the effect of acetylation on the multiple extra mitochondrial functions of Cyt c and its implication for the development of certain diseases. Gonzalo Pérez Mejias 68 3.4. Article IV: Electric field-induced functional changes in electrodeimmobilized mutant species of human cytochrome c In Olloqui-Sariego et al. (2022), we analyzed the thermodynamics and kinetics of electron exchange of surface-immobilized Cyt c variants under different local electric fields. In particular, we assessed the effect of Tyr48 and Tyr97 phosphorylation, as well as that of the naturally occurring thrombocytopenia-associated Tyr-to-His mutation at position 48, on the redox properties of human Cyt c. Tyr phosphorylation was mimicked using the noncanonical amino acid p-carboxymethyl- L -phenylalanine pCMF. Electrostatic binding to negatively charged SAM-coated electrodes stabilizes the ferric form of Cyt c species, lowering their E 1/2 . This observation agrees with previous report for horse Cyt c immobilized onto similar SAMs (Oviedo-Rouco et al., 2022). The stabilization of the ferric form is higher in the presence of a stronger interfacial electric field. Moreover, the E 1/2 difference between weak and strong interfacial electric fields of the Tyr48 and Tyr97 variants are doubled compared to WT protein. Such results suggest a clear influence of the local electric field on the modulation of the redox properties of immobilized human Cyt c. Analysis of the thermodynamic parameters associated with the heme reduction revealed that the enthalpic effects prevail over the entropic effects for all Cyt c species studied. The highly negative value of the standard enthalpy of reduction arises from the nature of the heme group cavity which is highly hydrophobic (Battistuzzi et al., 1997). Strikingly, MD simulation analyses revealed that there is a correlation between the decrease in the E 1/2 value for the Cyt c variants with the increase in the heme pocket surface and the reduction of the frequency of hydrogen bonds that involve residues neighboring the heme group (Figure 18). Results and Discussion 69 Figure 18. Molecular dynamics simulations of cytochrome c variants. Heme pocket surface (left) and frequency of heme-surrounding hydrogen bonds (right) of reduced Cyt c species calculated from MD simulations. The different Cyt c species immobilized under low electrostatic field displayed a similar temperature dependence of their electron transfer rate constants, yielding similar activation enthalpies associated with the electron transfer rate. However, Cyt c variants showed remarkably high values for the activation enthalpy and pre-exponential factors under strong interfacial electric field. The activation enthalpy is related to the reorganization energy (λ) of Cyt c, which can be defined as the sum of the inner and outer sphere contributions. The reorganization energy of the inner sphere (λ in ) is determined by the geometrical rearrangement of the redox active site accompanying the redox conversion. On the other hand, the reorganization energy of the outer sphere (λ out ) is associated with changes in the polypeptide environment of the redox site (λ p ) and solvent rearrangement (λ s ) following the ET step. In the case of human Cyt c, there are subtle differences in the heme group environment as evidenced by the solution NMR structure of reduced and oxidized Cyt c (Imai et al., 2016). The λ values obtained with immobilized proteins illustrate mainly the effect of λ in and λ p , as the contributions of λ s to Gonzalo Pérez Mejias 70 λ out are lower when the protein is immobilized on the surface (Chattopadhyay et al., 2021). An increase of the λ value for immobilized Cyt c has been previously associated with protein unfolding by immobilization with highly hydrophobic SAMs or urea treatment (Monari et al., 2010; 2011; Ranieri et al., 2015). However, the structural rearrangements induced by such conditions— e.g., major structural changes or detachment/exchange of the Met iron axial ligand—produce a significant shift of the E 1/2 by ca. 0.35 V. As the E 1/2 differences for herein studied Cyt c variants between weak and strong local interfacial electric field are in the 60-90 mV range, coarse structural changes or replacement of the axial ligand Met80 can be excluded. Therefore, the increase of the activation enthalpy and reorganization energy could be explained by subtle changes in the redox active site, induced by the alteration of the hydrogen-bonding network and the electrostatics of the heme crevice (Figure 18 and Figure 19). Figure 19. Modulation of heme crevice electrostatics and atomic fluctuations of cytochrome c species. Electrostatics surface potential at 150 mM ionic strength (left) and atomic fluctuations from MD simulations (RMSF, right) of Cyt c variants. Results and Discussion 71 Several studies performed with different c-type cytochromes have established the connection between the heme-CXXCH motif vibrational dynamics, the heme ruffling distortion, and the regulation of E 1/2 (Liptak et al., 2010; Galinato et al., 2012; 2015; Sun et al., 2014). Atomic fluctuations analyses from MD trajectories showed that the dynamics of the region corresponding to residues 10-30 are altered by mutations at positions 48 and 97 (Figure 19). Such differences might affect the heme-CXXCH vibrational dynamics upon Cyt c binding event, explaining the observed switch between low and high activation enthalpy. These results reveal a synergistic effect of the strong electrostatic adsorption during Cyt c binding and the naturally occurring tyrosine modification as responsible for their redox functional changes. This suggests that the modulation of the redox properties of the Cyt c variants arises from a subtle distortion of the active site, likely induced by the alteration of the hydrogen-bonding network and electrostatics of the heme crevice rather than by coarse structural changes, such as unfolding. 3.5. Article V: Phosphorylation disrupts long-distance electron transport in cytochrome c In Gomila, Pérez-Mejías, Nin-Hill et al. (2022), we investigate the molecular mechanism of ET regulation by Tyr48 phosphorylation in the Cyt c 1 - Cyt c complex. As described in Pérez-Mejías et al. (2020a), the physical contact between Cyt c 1 and Cyt c enhances the thermodynamic driving force for ET. However, the small E 1/2 difference between Cyt c 1 and Cyt c in an aqueous solution is compatible with a thermodynamically favored ET without requiring physical contact between the proteins. Indeed, it has been recently demonstrated that ET could take place between the soluble domain of Cyt c 1 and Cyt c across a phosphate-buffered aqueous medium over long distances (Lagunas et al., 2018). Such ET would occur without the need of establishing a well-defined complex, thereby reconciling weak binding with high Gonzalo Pérez Mejias 72 specificity, and maintaining a high turnover rate. The observation of longdistance inter-protein ET between Cyt c 1 and Cyt c has been explained by the presence of a charge conduit (Gouy-Chapman conduit) between both proteins (Lagunas et al., 2018). We combined single-molecule techniques, such as electrochemical scanning tunneling microscopy (EC-STM) and atomic force microscopy (AFM), biophysical protein-protein interaction techniques (SPR), enzymatic assays and computational calculations to characterize the effect of Cyt c Tyr48 phosphorylation on the ET process. Immobilization of Cyt c and Cyt c 1 on the probe and sample electrodes of EC-STM, respectively, allowed us to independently control the redox state of Cyt c 1 and Cyt c and compare the current-distance electrochemical decay of WT and Tyr48 phosphomimetic (modified with pCMF) Cyt c variant. Contrary to the Cyt c 1 -Cyt c system whose current decay spans several nanometers (Lagunas et al., 2018), experiments with the phosphomimetic variant revealed that current decay distances are halved, and the electrochemical gating is impaired (Figure 20a). MD simulations showed that the negative charge introduced by the pCMF residue on the Cyt c surface prevents cation exclusion between the proteins’ redox active sites, thereby enabling charge screening in this confined interface and disrupting the Gouy-Chapman conduit (Figure 20b). Cation depletion near the redox active sites allows the electric field to extend several nanometers, supporting the long-range ET (Lagunas et al., 2018). Therefore, the disruption of the Gouy-Chapman conduit evidence by MD simulations agrees with the EC-STM results, in which phosphorylation of Cyt c impairs the electrochemically gated long-range ET. Results and Discussion 73 Figure 20. Long-distance electron transfer between cytochrome c 1 and cytochrome c is impaired by cytochrome c Tyr48 phosphorylation. A) Currentdistance electrochemical tunneling spectroscopy results showing the ensemble of semilogarithmic current-distance curves during probe retraction experiments (left), the histograms of distance decay factors (β) calculate from individual curves (middle), and the average β values (right) for Cyt c 1 -Cyt c (red) and Cyt c 1 -Y48pCMF Cyt c (blue). B) Side view of averaged sodium concentration map (top) and computed electrostatic potential (bottom) calculated based on MD simulations of Cyt c 1 and Cyt c (WT and Y48pCMF) separated 4.2 nm (Fe-to-Fe distance). Average sodium concentration (right) over MD simulation within the area between both heme groups of Cyt c 1 and Cyt c (WT in red and Y48pCMF in blue). We further investigated the effect of phosphorylation in the interprotein interaction using single molecule and bulk measurements. Cyt c 1 - Y48pCMF Cyt c exhibits higher unbinding forces—measured by single molecule force spectroscopy with an AFM—than those of the complex with Gonzalo Pérez Mejías 80 5. Cytochrome c phosphorylation at Tyr48 impairs the electrochemically-gated long-range electron transfer from cytochrome c 1 by weaking the charge conduit between the partners. Stronger contact of phosphorylated cytochrome c with cytochrome c 1 (higher unbinding force and lower dissociation constant) disturbs the reversible binding found in equilibrium conditions, reducing the turnover of cytochrome c molecules at complex III. 81 CHAPTER 5 OUTLOOK AND FUTURE PERSPECTIVES “When you see a good move, look for a better one.” — Emanuel Lasker Outlook and Future Perspectives 83 CHAPTER 5: OUTLOOK AND FUTURE PERSPECTIVES In summary, this PhD thesis has provided new insights into the electron transfer of the human cytochrome c 1 -cytochorme c complex and into the regulation exerted by post-translation modifications in the mitochondrial cytochrome c functioning. Moreover, the research has highlighted the usefulness of molecular dynamics simulations as a decision-making tool for designing phosphomimetic variants. The three main structural techniques used in biology are X-ray crystallography, NMR and cryo-EM. While crystallographic studies allow to obtain single and well-defined complexes, NMR and cryo-EM, in conjunction with in silico calculations, provide an ensemble of conformations of the protein complexes. As most of the proteins perform their in vivo activity within hundreds of milliseconds, trying to understand how proteins function using individual static atomic-level structures can be challenging. X-ray crystallography depends on obtaining an ordered crystal, which is one of its major disadvantages. The structure present in the crystal represents only one of the possible conformations of the complex—the most stable one in the crystallization conditions—losing all information about the macromolecular dynamics occurring in the cell. For such reason, NMR and cryo-EM can be exploited to study multiple flexible conformations from experimental data. The advent of computational methods based on neural networks and artificial intelligence has revolutionized the field of structural biology, allowing for accurate prediction of 3D protein structures from their amino acid sequence (Jones and Thornton, 2022). However, the obtained models from such neural networks e.g., AlphaFold2 (Jumper et al., 2021), lack from information about dynamic properties of the protein structure or cannot properly predict the effect of genetic variants or specific post-translational modifications (Diwan et al., 2021). Gonzalo Pérez Mejias 84 By integrating the experimental data and the information obtained from computational calculations—combining MD methods with AlphaFold2 predictions, it is possible to achieve a more holistic understanding of biomolecular systems. This understanding could facilitate the development of new drugs and disease-fighting approaches. The fundamental principle of energy metabolism in biology states that the electron transfer between proteins occurs through direct interaction, with consequent coupling between their redox centers. Electronic coupling, or the strength of the interaction between electron-donating and electron-accepting redox centers, plays a crucial role in determining the speed and efficiency of long-range electron transfer. This coupling is typically stronger when the redox centers are close to one another, allowing for quick and efficient transfer. However, in certain cases, redox centers that are further apart can still have strong coupling, usually when there is a specific electron transfer pathway, such as through a protein or environment that facilitates the transfer over a longer distance. In our system (Cyt c 1 -Cyt c complex), we proposed that the long-range electron transfer between the protein partners is sustained by a Gouy-Chapman conduit (Lagunas et al., 2018; Gomila, Pérez-Mejias, Nin-Hill et al., 2022). This conduit refers to the electrostatic field that is generated by the charge distribution at the interface of a charged surface and an electrolyte solution. Specifically, when a charged surface is in contact with an electrolyte solution, the charge on the surface attracts counterions from the solution, creating a layer of positive and negative ions (the "diffuse double layer") near the surface. The charge separation in the diffuse double layer generates an electrostatic field that extends into the solution, enabling the transfer of electrons over long distances. This mechanism may be a general method for long-range electron transfer between protein complexes, as it has been observed a similar electrostatic distribution in Photosystem I from Arabidopsis thaliana, providing a favorable pathway for charge transport (López-Martínez et al., 2019). Outlook and Future Perspectives 85 Our findings have shown that Cyt c tyrosine 48 phosphorylation can shut down the ability of proteins to transfer electrons over long distances in aqueous solution by molding the ionic distribution around Cyt c 1 and Cyt c. Future research could investigate the effects of phosphorylation at other sites in Cyt c and other post-translational modifications—i.e., acetylation and nitration—and how this impacts the long-range electron transfer process. This would help to determine whether the proposed mechanism of regulation is specific to tyrosine 48 phosphorylation or a general mechanism for regulating Cyt c function. Moreover, it is intriguing to consider that the enhancement in the redox potential difference caused by the proximity of the partners—rather than direct contact between them—may lead to an optimal balance between the rate of electron transfer and turnover (Pérez-Mejías et al., 2020a). This opens up new possibilities for understanding the mechanisms of electron transfer, where the idea of a single, well-defined, close-range orientation is replaced by the concept of multiple and dynamic long-distance conformational states that are compatible with electron transfer. The specificity of the electron transfer will be sustained by the structural and chemical properties of the binding partners, as well as the redox potential difference. Another open question is whether the long-range electron transfer process could take place between Cyt c and complex IV. Recent studies have suggested the possibility, as the structure of the Cyt c-CIV complex in mammals contains three water molecule layers in the long space between the proteins, and a small contact surface, which could facilitate such a process (Shimada et al., 2017). Additionally, the main binding site for Cyt c in CIV is characterized by negatively charged residues similar to those found in the Cyt c-CIII binding sites. However, the CIV redox center that receives the electron from Cyt c is a binuclear copper cofactor, rather than a heme group (as in Cyt c 1 of CIII). For such reason, it would be interesting to study if it is possible the long-range electron transfer when the electron flows from Cyt c heme group to CIV binuclear copper cofactor. Gonzalo Pérez Mejias 86 It has been recently proposed that Cyt c may also have a role in the DDR mechanism by interacting with histone chaperones ANP32B and SET/TAF-Iβ (González-Arzola et al., 2015; Rivero-Rodríguez et al., 2021; Casado-Combreras et al., 2022). Moreover, as reported in González-Arzola et al., (2022), Cyt c translocates into the nucleolus on DNA insults, interacting with NPM1. NPM1 plays a key role in the formation of the nucleolus through liquid-liquid phase separation (LLPS) and contributes to the liquid-like features of the nucleolus (Mitrea et al., 2016; 2018). LLPS is a process in which specific regions of the cytoplasm or nucleoplasm can separate into distinct liquid droplets, named membraneless organelles, which can compartmentalize biochemical reactions and perform specific functions. Some examples of membraneless organelles are stress granules, P bodies, paraspeckles or the nucleolus. In recent years, there has been growing interest in the role of LLPS in biology, particularly in the context of disease. In some cases, misregulation of LLPS has been linked to the development of diseases such as cancer, neurodegeneration, and viral infection (Zbinden et al., 2020; Portz et al., 2021; Rai et al., 2021). The formation of these membraneless organelles is determined by unique molecular grammars which vary between the different types of condensates (Wang et al., 2018; Greig et al., 2020; Bremer et al., 2022; Ruff et al., 2022). Physicists, chemists, and biologists working on this research field have been trying to understand these molecular grammars by dividing protein sequences into cohesive “stickers” and solubility-influencing “spacers”. Different distribution of “stickers” and “spacers” as well as different composition of them lead to different types of condensates. The balance between NPM1 homotypic and heterotypic interactions is crucial for maintaining the structure of the nucleolus. Lysines of Cyt c are key residues involved in NPM1 binding and Cyt c acts as modulator of NPM1 conformational equilibrium as well as its multi-valency which affects the LLPS mechanism (González-Arzola et al., 2022). Outlook and Future Perspectives 87 An unexplored research area is the study of the regulation of Cyt c nuclear and nucleolar role by post-translational modifications. Recent research has shown that certain acidic regions of proteins, such as those of SET/TAF-Iβ, may function as "readers" for unacetylated forms of p53 protein. According to the authors of these studies (Wang et al., 2016; 2017), these "readers" of the unacetylated p53 C-terminal domain may also have the ability to recognize other lysine-rich regions in proteins, with Cyt c being a particularly noteworthy candidate due to its basic and lysine-rich properties. Moreover, authors have been able to generate facsimiles of core nucleolar substructures found in cells by phase separation of key proteins and nucleic acid mixtures (King et al., 2022). In this novel study, they have highlighted the role of lysine-rich domains and Asp and Glu repeat-rich tracts in the formation of the different phases/regions within the nucleolus, i.e., fibrillar center, dense fibrillar component and granular component. Based on the nature of the interactions between Cyt c and its nuclear/nucleolar partners, post-translational modifications are expected to have an impact on binding, specifically those affecting lysine residues. 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Scientific Articles 129 APPENDIX I: SCIENTIFIC ARTICLES Article I: Physical contact between cytochrome c 1 and cytochrome c increases the driving force for electron transfer  Pérez-Mejías G, Olloqui-Sariego JL, Guerra-Castellano A, Díaz- Quintana A, Calvente JJ, Andreu R, De la Rosa MA, Díaz-Moreno I. 2020. “Physical contact between cytochrome c 1 and cytochrome c increases the driving force for electron transfer”. Biochim. Biophys. Acta - Bioenerg., 1861: 148277. Contents lists available at ScienceDirect BBA - Bioenergetics journal homepage: www.elsevier.com/locate/bbabio Physical contact between cytochrome c 1 and cytochrome c increases the driving force for electron transfer Gonzalo Pérez-Mejías a , José Luis Olloqui-Sariego b,⁎ , Alejandra Guerra-Castellano a , Antonio Díaz-Quintana a , Juan José Calvente b , Rafael Andreu b , Miguel A. De la Rosa a , Irene Díaz-Moreno a,⁎ a Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de la Cartuja (cicCartuja), Universidad de Sevilla – Consejo Superior de Investigaciones Científicas (CSIC), Avda. Américo Vespucio 49, Sevilla 41092, Spain b Departamento de Química Física, Universidad de Sevilla, C/Profesor García González, s/n, Sevilla 41012, Spain ARTICLE INFO Keywords: Cytochrome c Cytochrome c 1 Complex III Cyclic voltammetry Heterogeneous electron transfer Nuclear Magnetic Resonance ABSTRACT In oxidative phosphorylation, the transfer of electrons from reduced cofactors to molecular oxygen via the electron transport chain (ETC) sustains the electrochemical transmembrane potential needed for ATP synthesis. A key component of the ETC is complex III (CIII, cytochrome bc 1 ), which transfers electrons from reduced ubiquinone to soluble cytochrome c (Cc) coupled to proton translocation into the mitochondrial intermembrane space. One electron from every two donated by hydroquinone at site P is transferred to Cc via the Rieskecytochrome c 1 (Cc 1 ) pathway. According to recent structural analyses of CIII and its transitory complex with Cc, the interaction between the Rieske subunit and Cc 1 switches intermittently during CIII activity. However, the electrochemical properties of Cc 1 and their function as a wire between Rieske and Cc are rather unexplored. Here, temperature variable cyclic voltammetry provides novel data on the thermodynamics and kinetics of interfacial electron transfer of immobilized Cc 1 . Findings reveal that Cc 1 displays two channels for electron exchange, with a remarkably fast heterogeneous electron transfer rate. Furthermore, the electrochemical properties are strongly modulated by the binding mode of the protein. Additionally, we show that electron transfer from Cc 1 to Cc is thermodynamically favored in the immobilized Cc 1 -Cc complex. Nuclear Magnetic Resonance, HADDOCK, and Surface Plasmon Resonance experiments provide further structural and functional data of the Cc 1 -Cc complex. Our data supports the Rieske-Cc 1 -Cc pathway acting as a unilateral switch thyristor in which redox potential modulation through protein-protein contacts are complemented with the relay-like Rieske behavior. 1. Introduction Mitochondria are fundamental organelles for the homeostasis of eukaryotic cells. In addition to being the main energy transducer, they play key roles in many cellular processes [1–3]. Core metabolic reactions take place within mitochondria, including among others the Krebs cycle, β-lipid oxidation and oxidative phosphorylation. The latter is catalyzed by the electron transport chain (ETC), which oxidizes small molecules like NADH or FADH 2 to reduce oxygen into water. This process is coupled with the translocation of protons from the mitochondrial matrix to the intermembrane space. Then, the resulting electrochemical gradient drives the synthesis of ATP by ATP synthase. Therefore, the proper performance and regulation of ETC is essential for cell survival and energy transduction. In fact, defaults in ETC https://doi.org/10.1016/j.bbabio.2020.148277 Received 30 May 2020; Received in revised form 29 June 2020; Accepted 9 July 2020 Abbreviations: AIRs, Ambiguous interaction restraints; BD, Brownian dynamics; Cc, Cytochrome c; Cc 1 , Cytochrome c 1 ; CI, Complex I; CII, Complex II; CIII, Complex III; CIV, Complex IV; CD, Circular dichroism; CSP, Chemical-shift perturbations; DLS, Dynamic light scattering; E 1/2 , Midpoint redox potential; ETC, Electron transport chain; FPLC, Fast protein liquid chromatography; ΔH rc0 , Standard enthalpy of reduction; ΔH ET⋕ , Activation enthalpy; HADDOCK, High ambiguity driven docking approach; HSQC, Heteronuclear single-quantum correlation; IMM, Inner mitochondrial membrane; ITC, Isothermal titration calorimetry; K D , Dissociation equilibrium constant; k ET0 , Heterogeneous standard electron transfer rate constant; k on , Association rate constant; k off , Dissociation rate constant; λ, Reorganization energy; MALDI-TOF, Matrix-assisted laser desorption ionization with time-of-flight; MD, Molecular dynamics; MPA, 3-mercaptopropionic acid; NMR, Nuclear Magnetic Resonance; PEG, Polyethylenglycol; RMSD, Root-mean-squared deviations; ROS, Reactive oxygen species; ΔS rc0 , Standard entropy of reduction; SAMs, Selfassembled monolayers; SPR, Surface Plasmon Resonance; TMAET, 2-(trimethylammonium)ethyl thiol; UV–Vis, Ultraviolet-visible; WT, Wild-type ⁎ Corresponding authors. E-mail addresses: [email protected] (J.L. Olloqui-Sariego), [email protected] (I. Díaz-Moreno). BBA - Bioenergetics 1861 (2020) 148277 Available online 24 July 2020 0005-2728/ © 2020 Elsevier B.V. All rights reserved. T components are implicated in a wide variety of diseases, as well as in the aging process itself [4–6]. The ETC comprises four protein complexes embedded in the inner mitochondrial membrane (IMM): NADH dehydrogenase or complex I (CI); succinate reductase or complex II (CII); cytochrome bc 1 or complex III (CIII) and cytochrome c oxidase or complex IV (CIV). Two soluble electron carriersenamely, quinone (Q) and cytochrome c (Cc)e are indeed responsible for transfer of electrons from CII to CIII and from CIII to CIV, respectively. As protons are pumped into the intermembrane space of the mitochondria, reactive oxygen species (ROS) may be generated. ROS are key molecules in many cellular processes such as aging and, paradoxically, produce critical signals for survival and cell proliferation [7–9]. CIII is one of the major sources of ROS generation, taking part in signal transduction into the mitochondria and seemingly participating in the development of certain types of cancer [10–12]. Mutations in the genes that encode various subunits of CIII have been related to a wide variety of diseases such as encephalopathies, lethal infantile liver failure, heart disease and various transitory episodes of hypoglycemia and lactic acidosis [13–17]. CIII is responsible for the transport of electrons from the Q pool to Cc (Fig. 1A). Functional CIII is a dimeric assembly located in the IMM (Fig. 1B). Each monomer has a molecular mass of 240 kDa and it is made up of 9–11 subunits [11,18]. The most important subunits are cytochrome b (Cb), Rieske protein and cytochrome c 1 (Cc 1 ), all of which are involved in the ETC [11]. The ETC in CIII begins with the oxidation of the quinones at the Q o site of Cb, where two electrons are transferred. One electron translocates to a Fe 2 S 2 center in the Rieske protein and subsequently to the heme group of Cc 1 , which ultimately donates it to Cc [11] (Fig. 1A). The other electron travels to the Q i site where it is transferred to an oxidized quinone molecule, generating a semiquinone species. This cycle is repeated again so the semiquinone is completely reduced to hydroquinone before dissociation. Cc 1 , the immediate electron donor to Cc, is an α-helix heme protein (ca. 27 kDa). It comprises two clearly differentiated regions: the N- terminal globular domain—located in the intermembrane space—and the C-terminal domain, a hydrophobic α-helix that anchors the protein to the IMM and the CIII core [19] (Fig. 1B). The N-terminal domain contains the heme c cofactor. An accepted model for the electron pathway through Cc 1 in a hypothetical human Rieske protein-Cc 1 -Cc complex is sketched in Fig. 1C [20]. This diagram illustrates that the electron is transferred into Cc 1 across the heme propionate side (channel I) and out of Cc 1 from a site next to the vinyl-group at ring C of the heme group (channel II). Initially, the Rieske protein head domain moves between the Q o site position and the Cc 1 position to donate the Fig. 1. Electron transport through the cytochrome c 1 subunit. A) Schematic representation of the electron flow (arrows) across the electron transport chain. Complex I (CI, NADH-ubiquinone oxidoreductase) is represented in yellow, complex II (CII, succinate-ubiquinone reductase) in blue, complex III (CIII, cytochrome bc 1 complex) in green, complex IV (CIV, cytochrome c oxidase) in purple, cytochrome c (Cc) in red and ubiquinone, as Q. IMS stands for intermembrane mitochondrial space, IMM for inner mitochondrial membrane and MM for mitochondrial matrix. The following redox cofactors are highlighted: CIII Rieske Fe 2 S 2 cluster (FeS, blue), c-type hemes (red for CIII cytochrome c 1 (Cc 1 ) and yellow for soluble Cc), CIII cytochromes b H and b L (b H and b L , pink) and CVI CuA center (Cu A , dark purple). B) Ribbon representation of the cryo-EM model for the dimeric structure of human CIII (PDB: 5XTE [19]). Soluble domain of Cc 1 is colored in blue, transmembrane Cc 1 helix in yellow, heme group in red and Rieske protein in green. C) Sketch of the electron pathway between Rieske-Cc 1 -Cc complex. Close-up view of the highlighted region in panel B, displaying the soluble N-terminal domains of Cc 1 and Rieske protein, along with Cc. The three cysteines mutated in Cc 1 by serine are highlighted in purple. The c-type heme groups are colored in red. The electrons (e − ) are transferred from [Fe 2 S 2 ] cluster at the Rieske protein to the heme group of Cc 1 through channel I (green). Then, electrons are transferred to the heme group of Cc through channel II (blue). G. Pérez-Mejías, et al. BBA - Bioenergetics 1861 (2020) 148277 2 electron, and presents two interaction domains which have been extensively studied [11,18,21–27]. Next, Cc interacts with Cc 1 to receive the electron. The interaction between Cc 1 and its counterpart has been characterized in yeast, plant and bacterial systems [28–34]. A related point to consider is that, under stress stimuli, Cc is released into the cytosol and nucleus where it interacts with several targets conserved in humans and plants [35]. Moreover, its function is highly regulated by post-translational modifications such as phosphorylation and nitration [36–41]. In recent years, several cryo-EM structures of mammalian and human supercomplexes—stable associations of different respiratory complexes—have been resolved [19,42–44]. However, these structures lack Cc molecules, and the mechanistic insights of the human Cc 1 -Cc complex remain unknown. Given that Cc 1 works at the IMM boundary, it is expected that interfacial interactions, such as protein-protein and protein-lipid interactions, and the hydrophobicity of the protein surroundings affect the function of this protein. Electrochemical measurements of immobilized proteins onto electrodes (so called protein film voltammetry) are a facile and effective method for gaining valuable information on the kinetics and thermodynamics of interfacial electron transfer in redox proteins and to identify structural alterations of metal centers associated with this redox reaction. A common strategy consists of the immobilization of redox proteins on gold electrodes modified with a thiol self-assembled monolayer (SAM), where the solution exposed tail group can be varied to allow for different modes of protein binding. While the direct electrochemistry of Cc adsorbed onto thiol SAMs has been extensively investigated, to the best of our knowledge, only two papers on the electrochemistry of adsorbed Cc 1 have been published, possibly due to the difficulties associated with the isolation and purification of this protein [45,46]. The aim of this work is to characterize the physicochemical properties of the water-soluble domain of human Cc 1 and its molecular recognition by human Cc, both in solution and in an immobilized state. The combination of computational and biophysical approaches herein assayed has allowed us to determine the residues involved in the Cc 1 -Cc interaction, which shares common features with previously studied complexes [33,47]. Our voltammetric measurements revealed that the conformational flexibility of Cc 1 is crucial for modulating its redox properties. Control of the redox potential of Cc and Cc 1 through this mechanism combined with the relay-like Rieske behavior may hamper reverse transfer of electrons. 2. Material and methods 2.1. Site-directed mutagenesis, protein expression and purification of recombinant proteins To express the soluble N-terminal domain of Cc 1 with three mutated cysteine residues, a synthetic DNA fragment encoding the soluble domain of the human C55S/C135S/C187S Cc 1 subunit (amino acids 85- 286 GenBank ID: 1537) was cloned into a pELB plasmid (provided by Prof. Inés Pereira at the Institute of Chemical and Biological Technology (ITQB), University of Lisbon). The pELB plasmid contains a leader sequence that targets the protein into the periplasmic space, thus minimizing its aggregation and degradation by proteases [48]. Escherichia coli (E. coli) BL21 (DE3) cells were co-transformed with the pELB-Cc1 and pEC86 plasmids—which contained the maturation and integration machinery of the heme group [49]. Cells were cultured for 20 h at 30 °C and 150 rpm in Luria-Bertani (LB) rich media supplemented with 0.1 mM δ-aminolevulinic acid, 0.1 mg mL −1 ampicillin and 0.02 mg mL −1 chloramphenicol. Cells were collected by centrifugation (9000 ×g for 10 min), and then suspended in lysis buffer (100 mM Tris- HCl, pH 8.0, supplemented with 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride and 0.02 mg mL −1 DNase). The periplasmic fraction was obtained by freeze-thaw lysis method. The resulting suspension was centrifuged at 20000 ×g for 30 min. The purification was carried out by affinity chromatography using a Fast Protein Liquid Chromatography (FPLC) system with a biotin matrix (StrepTrap HP 5 mL, GE Healthcare). The different fractions were eluted from the column with 2.5 mM desthiobiotin in binding buffer and tested by ultraviolet-visible (UV–Vis) spectrophotometry in a Jasco V-630 apparatus. The A 280 /A 552 ratio of the resulting Cc 1 preparations in the reduced state was ca. 2.0. Tryptic digestion and MALDI-TOF analysis confirmed the molecular mass and the different mutations. Protein concentrations were determined by visible spectrophotometry, using an extinction coefficient of 28 mM −1 cm −1 at 552 nm wavelength (ε 552nm ) for reduced Cc 1 [33]. The oligomerization state of recombinant Cc 1 was assessed by dynamic light scattering (DLS) using a Zetasizer Nano ZS equipment (Malvern). To obtain wild-type (WT) Cc species, we proceeded as previously described with the E. coli BL21 (DE3) strain using the pBTR-1 plasmid [50]. This plasmid comprises the CYCS and CYC3 genes, coding for human Cc and the yeast Cc heme lyase, needed for correct heme integration in c-type cytochromes [51]. Tryptic digestion and MALDI-TOF analysis confirmed the molecular mass. Protein concentration was determined by visible spectrophotometry, using an extinction coefficient (ε 550nm ) of 29 mM −1 cm −1 for reduced Cc [50]. 2.2. Electronic absorption spectroscopy The colorimetric properties of the soluble domain of Cc 1 were analyzed by monitoring the changes in absorption maxima, in particular the 552-nm band. Absorption spectra were recorded in the UV–Vis range at 298 K, using a Jasco® V-650 spectrophotometer and a 10 mm path-length quartz cuvette. Samples contained 0.1 mM of either oxidized or reduced Cc 1 in 10 mM sodium phosphate buffer, pH 6.5, supplemented with 1 mM potassium ferricyanide or 1 mM sodium ascorbate, respectively. 2.3. Circular dichroism spectroscopy Secondary structure analysis of Cc 1 was performed by circular dichroism (CD). Spectra were recorded in the UV range (190–250 nm) at 298 K using a Jasco® J-815 spectropolarimeter equipped with a Peltier temperature-control system. CD intensities are presented in terms of molar ellipticity [θ molar ] using molar protein concentration. Sample contained 3 μM protein in 10 mM sodium phosphate buffer, pH 6.5, supplemented with 10 μM potassium ferricyanide to keep the oxidized state of Cc 1 . For each sample, 20 scans were averaged. The environment of the Cc 1 heme group was monitored by recording visible CD spectra (300–600 nm) at 298 K in a 10-mm pathlength quartz cuvette. Sample contained 30 μM protein in 10 mM sodium phosphate buffer, pH 6.5, supplemented with 100 μM potassium ferricyanide. 2.4. Nuclear Magnetic Resonance experiments 1D 1 H Nuclear Magnetic Resonance (NMR) spectra of reduced Cc 1 was performed on a 700 MHz Bruker Avance-III spectrometer equipped with a cryoprobe. Water signal was suppressed by excitation sculpting method [52]. Protein concentration was 25 μM in 10 mM sodium phosphate buffer, pH 6.3. Ascorbic acid was added to ensure the redox state for the sample. NMR titrations of 25 μM of 15 N labeled Cc with aliquots of unlabeled Cc 1 were performed in 10 mM sodium phosphate buffer, pH 6.3, and 10% D 2 O. The pH of the samples was checked before and after recording every spectrum. Either ascorbic acid or potassium ferricyanide was added to maintain the redox state of each sample. The chemicalshift changes were monitored in a series of 2D [ 1 H, 15 N] HSQC experiments at 298 K and recorded on a Bruker Avance-III 700 MHz equipped with a cryoprobe. NMR assignments of the 15 N and 1 H nuclei of reduced Cc (Cc red , BMRB accession number 25907) and oxidized Cc G. Pérez-Mejías, et al. BBA - Bioenergetics 1861 (2020) 148277 3 (Cc oxd , BMRB accession number 25908) were taken from previous work [53]. The data were processed using Bruker TopSpin NMR 4.0.4 software (Bruker), and chemical-shit perturbation (CSP) was analyzed with NMRFAM-Sparky NMR assignment tool [54]. 2.5. NMR-driven docking Restrained docking calculations were performed with the High Ambiguity Driven Docking approach (HADDOCK) [55,56]. Cc (PDB: 2N9I [53]) and Cc 1 (PDB: 5XTE [19]) structures were used as input models. Cc residues with CSP > 2σ were set as active residues. These active residues were Lys7, Phe10, Ile11, Met12, Lys13, Cys14, Gln16, Cys17, Val20, Ser47, Ala50, Ala51, Lys72, Lys73, Ile81, Val83 and Glu89. Cc residues exposed to solvent and located at < 4 Å from the active ones were labeled as passive residues. One active residue of Cc 1 was defined (Met160) and passive residues were those with a high solvent accessibility (> 50%) calculated with NACCESS [57]. These residues were Gln35, Gln71, Pro74, Glu76, Asp77, Glu79, Pro96, Ala103, Ala104, Asn106, His121, Glu136, Thr139, Gly140, Ser142, Arg144, Glu145, Asp167, Glu170, Asp172, Gly174 and Pro176. Ambiguous interaction restraints (AIRs) for the docking simulation were generated using standard criteria. Random exclusion of AIRs was set to 2%. For each run, 5000 rigid-body solutions were generated by energy minimization. The 400 structures with lowest AIRs energies were subjected to semi-flexible simulated annealing. Then, the first 200 structures were submitted to a final refinement in explicit water. The structures were analyzed using the root-mean-squared deviations (RMSD) clustering method with 7.5 Å cutoff. Clusters were classified according to HADDOCK score and representative structures were analyzed using UCSF Chimera [58]. Centers of mass were calculated as previously reported [59]. 2.6. Surface Plasmon Resonance Analysis of Cc binding to Cc 1 was performed by Surface Plasmon Resonance (SPR) using a CS SPRi-Biochip™ (HORIBA Scientific) and a SPRi-Plex II™ system (HORIBA Scientific). Cc 1 was covalently attached to the matrix using CS direct amine-coupling chemistry, and carried out by deposition of 10 and 15 μM solution drops on the surface. Rabbit serum albumin was attached to the chip surface at reference spots used as a control. The binding assays were performed at 298 K in 10 mM sodium phosphate buffer, pH 6.5. Cc at various concentrations (from 2.5 to 10 μM) were flowed on the surface at a 50 μL min −1 rate. The signals from the reference spot surface were subtracted in every sensogram. The data were analyzed with Origin 2018 (OriginLab Corporation). 2.7. Electron transfer pathway calculation Electron transfer pathway in protein complex was calculated with eMap [60]. Distance option calculation was set to closest atom, and distance cut-off was 20 Å. Trp, Tyr, Phe and His residues were included in the analysis. Electron source was set to Fe 2 S 2 cluster of Rieske protein and electron target to heme group of Cc. 2.8. Electrochemical measurements analysis For the electrode modification, ω-mercaptocarboxylic acid (HS- (CH 2 ) n -COOH) with n = 2, 3, 6, 8, 11, i. e. 2-mercaptoacetic acid, 3- mercaptopropionic acid, 6-mercaptohexanoic acid, 8-mercaptooctanoic acid and 11-mercaptoundecanoic acid, 1-propanethiol (S-(CH 2 ) 2 -CH 3 ) and 6-mercaptohexanol (S-(CH 2 ) 5 -CH 2 OH) (from Sigma Aldrich) were used without further purification. 2-(trimethylammonium)ethyl thiol was synthesized by acid-catalyzed hydrolysis (with aqueous methanolic hydrochloric acid) of the acetylcholine chloride (≥99% from Sigma Aldrich) and used without further purification. Buffer solutions were prepared from anhydrous sodium dihydrogen phosphate (from Fluka) and Millipore water. The viscosity of the solution was varied by adding a variable amount of a solution containing 35% w/w polyethylenglycol (PEG 8000, from Fluka) and 20 mM sodium phosphate buffer, pH 7.0. Solution viscosity values for different concentrations of PEG and temperatures were computed by using Gonzalez-Tello's equation [61]. Polycrystalline gold working electrode had a 0.0314 cm 2 geometric area. Prior to measurements, the gold surface was cleaned by successively polishing with 0.3 and 0.05 μm alumina and rinsing with Millipore water. Then, it was sonicated in absolute ethanol to remove residual alumina. The surface was subsequently dried and chemically cleaned using a “piranha” solution (7:3 mixture of concentrated H 2 SO 4 and 30% v/v H 2 O 2 ). SAMs were prepared by immersing the electrode into a 1 mM thiol-ethanolic solution for 30 min at room temperature. Protein immobilization was carried out for 90 min at 277 K, by deposition of a 10 μL drop of a solution containing 12 μM protein, 10 mM sodium phosphate buffer at pH 7.0 onto the modified electrode. Linear scan voltammetric measurements were performed with an AUTOLAB PGSTAT-30, from Eco Chemie B·V, in a three-electrode undivided glass cell, equipped with a gas inlet and thermostated with a water jacket. The counter and reference electrodes were a Pt bar and an Ag/AgCl/NaCl saturated electrode, respectively. The reference electrode was connected to the cell solution via a salt bridge, and kept at room temperature (298 ± 2 K) in a non-isothermal configuration. Reported potential values have been corrected to the NHE potential scale by adding +192 mV to the experimental potential values. All measurements were carried out under argon atmosphere. Working solutions contained 20 mM sodium phosphate buffer, pH 7.0. 3. Results and discussion 3.1. Expression of soluble N-terminal domain of Cc 1 allows the study of its biophysical properties To study the biophysical and redox properties of Cc 1 , we cloned the N-terminal water-soluble domain of Cc 1 . To achieve this, the protein sequence was truncated as in previous studies with soluble domains of Cc 1 from other sources [33,45,62–64]. The residues Ser1 to Arg201 from the mature physiological protein (Genbank 1537) were kept and the C-terminal hydrophobic helix was deleted (Fig. 1B). Additionally, cysteine residues 55, 135 and 187 were mutated to serine to avoid dimerization through intermolecular disulphide bridges (Fig. 1C). The resulting recombinant, site-directed mutated domain of Cc 1 was successfully expressed in E. coli and purified (Fig. S1A–B). The molecular mass of soluble Cc 1 was 24,314.9 Da, as determined by MALDI-TOF analysis, and DLS corroborated its monomeric state (Fig. S1C–D). Proper folding and heme coordination were evaluated by CD and NMR spectroscopies. Analysis of CD measurements on the far-UV region provided information on the secondary structure of soluble Cc 1 , which consisted mainly of α-helices (Fig. 2A). Similar measurements in the UV–Vis region were performed to evaluate the heme environment [65] (Fig. 2B). The spectra exhibited the characteristic signal profile of properly coordinated c-type heme groups and showed a large positive Cotton effect, typical of Cc 1 [66]. The 1D 1 H NMR spectrum displayed broad amide-signal dispersion, distinctive of three-dimensional folding (Fig. 2C). The up-field resonance at ca. –3 ppm corresponds to the ε-CH 3 group of Met160 and is consistent with its role as a heme-axial ligand. Notably, this signal undergoes slow exchange (t 1/2 ≫ 28 ms) with the secondary species being populated as the temperature increases. Nevertheless, the structural change in Met160 causing this effect is rather small, taking into account the small chemical-shift difference and the porphyrin ring current influence. In addition, the UV–Vis absorption spectrum of oxidized soluble Cc 1 displayed absorption maxima at 529 (Q band) and 411 nm (Soret band), which are characteristic of a low spin state in the heme iron. Upon reduction, the Soret band shifts to 417 nm, and the spectra shows α- and β-bands at 552 and 522 nm, G. Pérez-Mejías, et al. BBA - Bioenergetics 1861 (2020) 148277 4 respectively (Fig. S1E). This data is consistent with the correct incorporation of the heme group into the apoprotein, as previously reported [33,62,67,68]. 3.2. Voltammetric response of Cc 1 adsorbed on SAMs Redox properties were studied by performing cyclic voltammetric experiments on the interfacial electron transfer of Cc 1 adsorbed onto polycrystalline gold electrodes modified with thiol SAMs bearing different terminal groups. Specifically, gold electrodes modified with 6- mercaptohexanol (MHOL) were used to study the redox properties of Cc 1 adsorbed on a surface that mimics its interaction with water molecules. In fact, its voltammetric midpoint potential (E 1/2 ) was +230 mV vs. NHE (Fig. 2D), close to the value determined for photosynthetic bacterial and plant Cc 1 in solution (ca. +250 mV) [68–70]. Later, similar measurements were conducted on gold electrodes modified with 3-mercaptopropionic acid (MPA) and 2-(trimethylammonium)ethyl thiol (TMAET) SAMs, which provided negatively and positively charged surfaces respectively at physiological pH 7. Each monolayer was expected to drive protein binding in a different electrostatically-induced orientation. The electrostatic potential distribution of Cc 1 around channel II, where it donates electrons to Cc, is highly negative, so the use of the positively charged TMAET SAM was intended to mimic the interaction between Cc 1 and Cc. On the other hand, the area surrounding channel I, where it receives electrons from the Rieske protein, is positively charged (Fig. 3A), so the use of the negatively charged MPA SAM was intended to mimic the interaction between Cc 1 and the Rieske protein. Cyclic voltammograms were recorded at physiological pH 7 over a wide range of scan rates (from 0.02 to 500 V s −1 ) and temperatures (from 273 K to 323 K). Irrespective of the nature of the thiol monolayer, adsorbed Cc 1 showed a quasi-ideal voltammetric response for its Fe(III)/Fe(II) redox couple (Fig. 3B). Integration of the baseline-corrected voltammetric peaks provides protein coverage values of ca. 4 pmol cm −2 , which are close to a protein monolayer, as estimated from the crystallographic dimensions of the protein scaffold. The E 1/2 of Cc 1 adsorbed onto the MPA SAM is +134 mV vs. NHE, i.e. 90 mV more negative than the value obtained for Cc 1 adsorbed onto the MHOL SAM. This potential downshift of the electrostatically adsorbed protein can be attributed to the stabilization of the positively charged ferric form of the native heme upon immobilization on a negatively charged surface. According to the electrostatic potential distribution of Cc 1 (Fig. 3A), the more plausible scenario implies the binding of Cc 1 to the negatively charged MPA SAM via an attractive electrostatic interaction with the positive patch located near the entrance of channel I; a physical scenario similar to that observed in the Cc 1 –Rieske complex. In this conformation, heme propionates may be at least partially concealed from the solvent, and their ionization equilibria be affected by the negatively charged SAM surface, thereby impacting the redox potential of the heme group [71]. In this sense, the investigation of pH dependence of the midpoint potential in the physiological pH interval between 5.5 and 7.75 (Fig. S2), reveals that it varies linearly with pH, with a slope of ca. 60 mV/pH, thereby indicating that the interfacial electron transfer is coupled to a monoprotonic transfer. Under the proposed protein orientation on the MPA SAM, we can infer that the charge compensation that accompanies the electron transfer might occur via a proton uptake from the monolayer carboxylic groups by a charged residue readily accessible to the SAM and close to the heme. Fig. 2. Biophysical properties of the recombinant soluble domain of Cc 1 . A) Far-UV CD spectrum of oxidized Cc 1 . B) Visible CD spectrum of oxidized Cc 1 . C) 1D 1 H NMR spectrum of reduced Cc 1 . Inset shows how the chemical shift and exchange regime of the heme axial ligand Met160-εCH 3 signal changes at different temperatures. D) Raw and baseline cyclic voltammogram recorded at 0.1 V s −1 and 298 K of Cc 1 immobilized onto a gold electrode modified with 6-mercaptohexanol self-assembled monolayer (SAM). Buffer was 20 mM sodium phosphate, pH 7. G. Pérez-Mejías, et al. BBA - Bioenergetics 1861 (2020) 148277 5 However, when Cc 1 was adsorbed onto the positively charged TMAET SAM, E 1/2 shifts to −233 mV, and becomes independent of the solution pH (Fig. S2), suggesting a conformational change in the heme pocket of the protein upon physisorption on this positively charged surface. The large negatively charged path around channel II suggests that this is the docking area where the protein contacts the TMAET SAM, in an analogous manner to the formation of the Cc 1 -Cc complex. A similar E 1/2 downshift has been previously reported by Osyczka et al. [72] for a M183H mutant variant of bacterial cytochrome c 1 from Rhodobacter capsulatus, where the axial heme ligand methionine was replaced by a histidine residue [72–76]. Thus, one can tentatively assign this conformational change to a low-spin six-coordinated His/Hisligated heme non-native conformer. Moreover, the structure of this bishistidine conformer has been extensively investigated and characterized in its Cc counterpart, which displays redox changes similar to those we have observed in Cc 1 [77–81]. Unlike Cc, in Cc 1 all histidine residues lie far from the heme pocket, so a Met-to-His ligand exchange may be less probable in this case. Nevertheless, a local conformation change might still account for the potential downshift if one considers distinct but synergic effects [82]. For instance, strengthening the hydrogen bond between the carbonyl oxygen of Pro111 and Hδ1 His41 may increase the imidazolate character of the histidine ligand. This may decrease the redox potential as much as 100 mV. Moreover, opening of the non-polar heme cavity to the solvent—for instance, by a small displacement of helix IV, which separates the two channels—could cause a substantial decrease in redox potential. In fact, displacement of Ala108 methyl group would substantially increase the accessibility of porphyrin to the solvent. Nevertheless, such analysis lies beyond the scope of this work and will be addressed elsewhere. The more positive E 1/2 value (−150 mV) observed for free hemin adsorbed onto TMAET-modified gold electrodes (Fig. S3) discards the possibility of a high-spin five-coordinated heme conformational state, in which the methionine axial ligand is detached from the iron upon immobilization of Cc 1 on this SAM. Such an assumption is further supported by the observed upshift in the Cc 1 midpoint potential to a value similar to that of hemin after immersion of the Cc 1 /TMAET/Au electrode into a denaturing 9 M urea aqueous solution for 2 h, which is known to induce protein unfolding to a more extended and open protein structure (Fig. S3). Under these assumed scenarios that mimic either the Cc 1 -Cc or the Cc 1 -Rieske complex, and based on the voltammetric findings, one can hypothesize that in the Cc 1 -Cc complex, the Cc 1 protein undergoes a conformational change lowering its E 1/2 , thus making electron transfer from Cc 1 to Cc energetically favorable. Analysis of the crystallographic structure of yeast Cc 1 -Cc complex revealed considerable rearrangement of water molecules upon Cc binding to Cc 1 [47]. Additionally, fluctuations on the Cc 1 extended β-strand finger (residues 130–152) and the cluster of acidic residues Asp231, Asp232, Glu235, Glu237, and Asp238, and the neighboring Gly239 were observed in molecular dynamic simulations of the yeast Cc 1 -Cc complex [83]. Whereas, Cc 1 retains its native conformation in the Cc 1 –Rieske complex with a large redox potential gradient between the Rieske protein and Cc 1 thermodynamically favoring the electron transfer from Rieske to Cc 1 [84,85]. Overall, it seems that the local potential distribution across the electrode–SAM–protein–electrolyte interface modulates the measured redox potentials which may be important for transient states or binding/unbinding events in the Cc 1 -counterpart complexes during its biological function. 3.3. Thermodynamics and kinetics of the interfacial electron transfer of immobilized Cc 1 To gain information on the influence of the binding mode on the kinetics and thermodynamics of the interfacial electron transfer of Cc 1 , we investigated the effect of temperature on the voltammetric response of immobilized Cc 1 . Fig. 3C shows the temperature dependence of E 1/2 for Cc 1 adsorbed on MPA and TMAET SAMs. In both cases, a linear decrease in E 1/2 upon increasing temperature was observed, the Fig. 3. Electrochemical characterization of the recombinant soluble domain of Cc 1 immobilized onto charged self-assembled monolayers. A) Electrostatic Surface Potential (ESP) of Cc 1 calculated at 150 mM ionic strength. Electron channels I and II are indicated. The heme group is colored in green. B) Voltammetry response—recorded at 5 V s −1 and 298 K—of Cc 1 immobilized onto a gold electrode modified with 3-mercaptopropionic acid (red) or 2-(trimethylammonium) ethyl thiol (blue) SAMs. C) Variation of the midpoint potential E 1/2 with temperature. D) Arrhenius plot of the standard electron transfer rate constant k ET of Cc 1 immobilized onto a gold electrode modified with 3-mercaptopropionic acid (red symbol) or 2-(trimethylammonium) ethyl thiol (blue symbol) SAMs. Solid lines are the best linear least-squares fits of the data. Buffer was 20 mM sodium phosphate, pH 7. G. Pérez-Mejías, et al. BBA - Bioenergetics 1861 (2020) 148277 6 decrease being steeper when Cc 1 was adsorbed on MPA. The two distinct dependences of E 1/2 support the presence of surface induced conformational changes that affect electron transfer energetics. The values of reduction entropy (ΔS rc0 ) and enthalpy (ΔH rc0 ) were estimated from the slopes of the E 1/2 vs. T plots (Fig. 3C), and are listed in Table 1. It turns out that the redox thermodynamics of Cc 1 adsorbed on MPA are characterized by negative ΔS rc0 and ΔH rc0 values, as described in previous results for mammalian Cc adsorbed on anionic surfaces, revealing large differences in the protein solvation and/or structure upon changing its oxidation state [86,87]. On the other hand, when Cc 1 was adsorbed on TMAET SAM, ΔH rc0 was positive, as found previously for other His/His-ligated forms of Cc [79]. This observation has been ascribed to the stabilization of the ferric heme group by the axial histidine residue, whereas the smaller absolute value of ΔS rc0 indicates that both solvation and conformational flexibility are largely unaffected by the electron transfer process. The effect of the monolayer nature on the kinetics of the electron exchange between Cc 1 and the electrode was also investigated from the variation of the midpoint potential with scan rate. As the scan rate is being increased, the two voltammetric peaks depart progressively from each other, producing trumpet plots (Fig. S4). The apparent standard electron transfer rate constant k ET0 values were determined from the trumpet plot with a fitting procedure by assuming the Butler-Volmer kinetic formalism with a transfer coefficient of 0.5, and with the addition of a small finite peak potential separation to account for the persistent low scan rate peak splitting (Fig. S4). Their values at 298 K are listed in Table 1. Irrespective of the SAM nature, nearly symmetrical trumpet plots are obtained, as expected for a well behaved and quasireversible one electron redox couple. However, two deviations from the ideal behavior are observed: i) a small variation of the average peak potential with scan rate, and ii) a persistent peak potential separation at low scan rate, commonly known in literature as unusual quasi-rever- sibility, which are ascribed to surface hysteresis effects [88] and/or a hysteresis produced by the shape of the reaction coordinate [89]. Notably, immobilized Cc 1 onto either negatively or positively charged SAMs, displays very high k ET0 values, suggesting a close contact between the heme center and the electrode, a finding which is in agreement with the proposed orientation adopted by Cc 1 upon adsorption on the different SAMs. The temperature dependence of k ET0 for Cc 1 was also investigated (Fig. 3D). Activation enthalpy values were obtained from the slopes of the linear Arrhenius plots and are listed in Table 1. Notably, electron transfer of Cc 1 immobilized onto MPA SAM proceeds with a significantly high ΔH ET⋕ value of 22.8 kJ mol −1 . Such a high ΔH ET⋕ value suggests a strong involvement of the solvent in the activation process, which may be explained by the existence of two opened heme cavities (see Fig. 3A), which are absent in Cc. When Cc 1 was adsorbed onto the TMEAT SAM, a lower ΔH ET⋕ value of 17.0 kJ mol −1 was obtained, in agreement with the smaller absolute variations of both entropy and enthalpy of reduction that were recorded with this monolayer. The lower absolute values for ΔH ET⋕ , ΔH rc0 and ΔS rc0 can be explained by the presence of hydrophobic interactions between the methyl substituents in the monolayer and the heme crevice opening, which diminish the water content in this region [90,91]. The extremely fast rate of electron exchange between Cc 1 and the electrode could be ascribed to strong electronic coupling between the heme group and the Table 1 Thermodynamic and kinetic parameters of the Fe(III)/Fe(II) redox conversion of immobilized human cytochrome c 1 (Cc 1 ) in 3-mercaptopropionic acid (MPA) or 2-(trimethylammonium)ethyl thiol (TMAET) self-assembled monolayers. Cc 1 /MPA Cc 1 /TMAET E 1/2 (mV vs. NHE) a 134 ± 5 −233 ± 5 ∆S rc0 (J K −1 mol −1 ) −71 ± 5 −17 ± 3 ∆H rc0 (kJ mol −1 ) −34.2 ± 1.4 17.6 ± 0.9 k ET0 (s −1 ) a 2800 ± 100 4400 ± 200 ∆H ET# (kJ mol −1 ) 22.8 ± 0.6 17.0 ± 0.8 a Measured at 298 K. Fig. 4. Thermodynamic characterization of the immobilized Cc 1 -Cc complex. Baseline-corrected and scan rate normalized cyclic voltammograms (upper) of Cc 1 (red), Cc (purple), and the Cc 1 -Cc complex (green) immobilized onto a gold electrode modified with a 8-mercaptooctanoic acid SAM, recorded at the indicated potential scan rate. Their corresponding midpoint potential variation (lower) with the logarithm of the scan rate (v). Buffer was 20 mM sodium phosphate pH 7; and temperature was 278 K. Table 2 Midpoint potentials of the Fe(III)/Fe(II) redox conversion of human cytochrome c 1 (Cc 1 ), cytochrome c (Cc) and cytochrome c 1 – cytochrome c (Cc 1 -Cc) sequential mixture immobilized onto 8-mercaptooctanoic acid self-assembled monolayers. Low scan rate (v = 0.01 V s −1 ) High scan rate (v = 10 V s −1 ) E 1/2 (mV vs. NHE) E 1/2 (mV vs. NHE) Cc 1 128 ± 5 135 ± 7 Cc 1 + Cc 211 ± 5 110 ± 7 Cc 178 ± 5 171 ± 5 G. Pérez-Mejías, et al. BBA - Bioenergetics 1861 (2020) 148277 7 1 Physical contact between cytochrome c1 and cytochrome c increases the driving force for electron transfer SUPPORTING INFORMATION Gonzalo Pérez-Mejías1, José Luis Olloqui-Sariego2,*, Alejandra Guerra-Castellano1, Antonio Díaz-Quintana1, Juan José Calvente2, Rafael Andreu2, Miguel A. De la Rosa1, Irene Díaz-Moreno1,* 1Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de la Cartuja (cicCartuja), Universidad de Sevilla – Consejo Superior de Investigaciones Científicas (CSIC), Avda. Américo Vespucio 49, Sevilla 41092, Spain. 2Departamento de Química Física, Universidad de Sevilla, C/ Profesor García González, s/n, Sevilla 41012, Spain. *Corresponding authors: [email protected] (J.O.S.); [email protected] (I.D.M.) Telephone number: +34 954 489513 Fax number: +34 954460165 2 Figure S1. Expression, purification and protein identification of recombinant soluble domain of cytochrome c1. A) 12% SDS-PAGE gel of the Cc1 soluble domain. 10 g of protein was loaded onto the gel. Gel was stained for protein detection with 0.25% Coomassie Brilliant Blue R-250 in 45% methanol and 10% acetic acid. M: Molecular weight marker. B) Trypsin digestion of Cc1 followed by matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) analysis. The expected tryptic masses clearly matched the calculated values, with 0.5 Da tolerance. The sequence coverage of these fragments is shaded in yellow. C) MALDI-TOF spectrum of Cc1. The calculated molecular mass of Cc1 is 24314.9 Da. D) Oligomerization state of Cc1. Average particle size distributions of Cc1 measured by dynamic light scattering (DLS). E) Visible absorption spectra of reduced (red) and oxidized (blue) Cc1. Soret (417 nm), − (552 nm) and -bands (522 nm) of reduced Cc1 are indicated. 3 Figure S2. Variation of the midpoint potential (E1/2) with pH of Cc1 immobilized onto a gold electrode modified with 3-mercaptopropionic acid (red) or 2‑(trimethylammonium) ethyl thiol (blue) SAMs at 298 K. Solid lines are linear least-square fits of the data. Buffer was 20 mM sodium phosphate. 4 Figure S3. Raw (left panel) and baseline corrected cyclic voltammograms (right panel) recorded at 5 V s-1 of Cc1 and hemin immobilized at 2-(trimethylammonium) ethyl thiol SAM (top panel), and Cc1 immobilized at 2-(trimethylammonium) ethyl thiol SAM before and after urea denaturation (bottom panel). Buffer was 20 mM sodium phosphate buffer solution pH 7 and temperature was 298 K. 5 Figure S4. Peak potentials (EP) as a function of the logarithm of the potential scan rate (v) for Cc1 immobilized onto a gold electrode modified with 3‑mercaptopropionic acid (red), 2‑(trimethylammonium) ethyl thiol (blue) and 6‑mercaptohexanol (black) SAMs. The solution pH 7 was controlled with a 20 mM sodium phosphate buffer and the temperature was 298 K. Solid lines are theoretical fits assuming the Butler-Volmer kinetic formalism with a transfer coefficient of 0.5, and addition of a small finite peak separation to account for the persistent low scan rate peak splitting. The fits provide the following values of the standard electron transfer rate constant: 2800 s-1 (red), 4400 s-1 (blue) and 25 s-1 (black). 6 Figure S5. Double logarithmic plot (top) of the overall standard electron transfer rate constant for Cc1 immobilized onto a gold electrode modified with 3-mercaptopropionic acid (red symbol) or 2-(trimethylammonium) ethyl thiol (blue symbol) SAM against solution viscosity at 298 K. Arrhenius plot (bottom) of the standard electron transfer rate constant of Cc1 immobilized onto a gold electrode modified with -mercaptocarboxylic acid SAM for the indicated number of methylene groups. Solid lines are the best linear least-squares fits of the data. Buffer was 20 mM sodium phosphate buffer solution pH 7. 7 Figure S6. Raw cyclic voltammograms of Cc1 (red) and Cc (purple), and of the Cc1-Cc complex (green) immobilized onto a gold electrode modified with a 8‑mercaptooctanoic acid SAM, recorded at the indicated potential scan rate. Buffer was 20 mM sodium phosphate, pH 7, and temperature was 278 K. 8 Figure S7. Peak potentials (EP) as a function of the logarithm of the potential scan rate (v) for Cc1 (red) and Cc (blue) immobilized onto a gold electrode modified with 8‑mercaptooctanoic acid SAM. Buffer was 20 mM sodium phosphate pH 7, and temperature was 278 K. Solid lines are theoretical fits assuming the Butler-Volmer kinetic formalism with a transfer coefficient of 0.5, and addition of a small finite peak separation to account for the persistent low scan rate peak splitting, providing the following values of the standard rate constant of electron transfer: 280 s-1 (red) and 320 s-1 (blue). 9 Figure S8. Midpoint potentials (E1/2) as a function of the logarithm of the potential scan rate (v) for Cc1 (red), Cc (purple), a preincubated mixture of Cc and Cc1 (light blue) and a sequential mixture of Cc plus Cc1 (yellow), all of them immobilized onto a gold electrode modified with 8‑mercaptooctanoic acid SAM. Buffer was 20 mM sodium phosphate, pH 7; temperature was 278 K. 10 Figure S9. Fits of Surface Plasmon Resonance curves. SPR response curves obtained with 5 M (yellow) and 2.5 M (blue) Cc analyte and distinct immobilized concentrations of Cc1 (left: 10 M; right: 15 M) are represented by triangles. Lines correspond to a simultaneous, global fit of the two curves to a one-site binding model. Data is representative of a total of 16 spots. 2 Supplementary Figure 1. Effect of tyrosine phosphorylation at position 48 on the structure of human cytochrome c. (Upper) Left: Superimposition of ribbon representations for wild-type Cc (red, PDB: 2N9I1) and its Y48pCMF variant (blue, PDB: 2N3Y2) in the reduced state. Location of Cys104—used for immobilization on Au(111) surface—is marked in yellow. Right: detailed view of the heme group, iron axial ligands (Met80 and His18) and Tyr48/pCMF48 residues, highlighting how phosphorylation induces drastic structural changes at the heme environment and tyrosine-harboring loop. (Lower) Surface representation of wild-type Cc (left, red) and its Y48pCMF variant (right, blue), with color ranging from lighter to darker for every residue depending on the intensity of their interaction with Cc1, as determined by NMR spectroscopy.2 The conformational adjustment induced by tyrosine phosphorylation enhances the heme group (green) solvent accessibility. The pictures were created with the UCFS Chimera 1.15rc software.3 3 Supplementary Figure 2. a, Schematics of the experimental set-up for current-distance (I-z) spectroscopy studies of cytochrome c (Cc) under bipotentiostatic control in near to physiological environment in EC-STM. Abbreviations: WE, working electrode; RE, reference electrode; CE, counter electrode. b, EC-STM image of 400x400 nm of Cc immobilized on Au(111) electrode through the C-terminal mutation E104C. Current set point = 0.3 nA. c, Cyclic voltammetry of Au(111) electrodes modified with Cc (orange) and Y48pCMF Cc (navy). The corresponding reduction (Uc) an oxidation (Ua) peaks for the Fe3+/Fe2+ couple are indicated. d, Ensemble of semilogarithmic current-distance (I-z) curves obtained during probe retraction (15 nm from the set point at 12 nm/s) for Cc (orange) and Y48pCMF Cc (navy). Current set point = 0.4 nA. e, Histograms of distance decay factors (β) quantified from individual curves in d, and the corresponding plot of the averaged β values from gaussian fit (mean ± s.d.) showing no statistically significant differences (two sample t-test, t Statistic = 1.111, degrees of freedom = 130.0, P < 0.05). β = 3 ± 1 nm-1 for both Cc (n = 75 I-z curves from 2 independent experiments) and Y48pCMF Cc (n = 82 I-z curves from 2 independent experiments). All the experiments were performed in 50 mM phosphate buffer, pH 6.5. Us = -200 mV and at constant bias of 800 mV. Source data are provided as a Source Data file. 4 Supplementary Figure 3. MD simulations side view of the average water concentration (M) map at the X-axe plane dissecting both Cc1-Cc redox active sites facing each other, set 3.2 nm apart taking the most external hydrogen atom from the -CH3 distal pairs from each heme group, that is 4.2 nm taking the Fe-to-Fe distance, in the aqueous medium, at the same plane as Figure 4 of the main text. (a) Cc1-Cc, (b) Cc1-Y48pCMF Cc in silico, (c), Cc1-pY48 Cc (O-phospho-L-tyrosine) with charge –1, (d) Cc1-pY48 Cc (O-phospho-L-tyrosine) with charge –2. Each contour line displays an increase of 50 units in concentration. No major differences in water concentration are observed and only a mild higher mobility of the phosphorylated Cc is perceived. Y48pCMF Cc displays a more accessible surface for water molecules due to a more flexible structure. (e, f) Side view of the averaged sodium concentration (M) map at the X-axe plane dissecting both Cc1 – Cc redox-active sites (pY48 Cc q-1 (e) and pY48 Cc q-2 (f), respectively) facing each other as described. Each contour line displays an increase of 0.15 M. Source data are provided as a Source Data file. 5 Supplementary Figure 4. Complex III activity with WT or Y48pCMF Cc as the electron acceptor. Activities were measured by monitoring the absorption changes at 550 nm and reported as mean ± s.d. (n=3 and 5 independent experiments for WT and Y48pCMF, respectively), showing significant differences (two sample t-test were used; t Statistic=6.22, degrees of freedom=6.00, ***P=4.00x10-4). Source data are provided as a Source Data file. Supplementary References 1. Imai, M. et al. Investigation of the redox-dependent modulation of structure and dynamics in human cytochrome c. Biochem. Biophys. Res. Commun. 469: 978–984 (2016). 2. Moreno-Beltrán, B. et al. Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48. Proc. Natl. Acad. Sci. 114: E3041-E3050 (2017). 3. Pettersen, E. F. et al. UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 25: 1605–1612 (2004). 261 APPENDIX II Review Articles 263 APPENDIX II: REVIEW ARTICLES Review I: Novel insights into the mechanism of electron transfer in mitochondrial cytochrome c  Pérez-Mejías G, Díaz-Quintana A, Guerra-Castellano A, Díaz-Moreno I, De la Rosa MA. 2022. “Novel insights into the mechanism of electron transfer in mitochondrial cytochrome c”. Coord. Chem. Rev. 450: 214233. Review Novel insights into the mechanism of electron transfer in mitochondrial cytochrome c Gonzalo Pérez-Mejías, Antonio Díaz-Quintana, Alejandra Guerra-Castellano, Irene Díaz-Moreno ⇑ , Miguel A. De la Rosa ⇑ Instituto de Investigaciones Químicas–cicCartuja, Universidad de Sevilla, Consejo Superior de Investigaciones Científicas (CSIC), Sevilla, Spain article info Article history: Received 1 June 2021 Received in revised form 24 August 2021 Accepted 19 September 2021 Available online 18 October 2021 Keywords: Cytochrome c Electron transfer Mitochondria Posttranslational modification Respiratory supercomplex abstract The supramolecular arrangement of respiratory complexes into supercomplexes is widely accepted. The common feature observed in the supercomplex architecture from organisms of diverse phylogenetic origin is the reduction of the distance between cytochrome bc 1 (complex III) and cytochrome coxidase (complex IV). Such an arrangement reduces the dimensionality (from 3D to 2D) of the diffusional search of cytochrome cas the electron carrier that connects both complexes. In this scenario, our recent finding of additional binding sites for cytochrome creinforces the concept of a ‘‘restrained 2D sliding pathway” onto the supercomplex surface. Herein, we analyze novel mechanistic insights into electron transfer towards cytochrome c, including modulation of the redox potential by physical contact as well as gated, long-range electron transfer through an aqueous solution. These data establish a new horizon for the understanding of electron transfer mechanisms beyond unique and well-orientated protein complexes. Multiple and dynamic long-distance conformational ensembles compatible with electron transfer could indeed contribute to the rapid adjustment of electron flow in response to changing cellular conditions. Ó2021 Elsevier B.V. All rights reserved. Contents 1. Introduction . . . ........................................................................................................ 1 2. Electron transport chain complexes . . . ..................................................................................... 2 3. Electron transport chain supercomplex organization . . . . . . . . .................................................................. 4 4. Novel insights into the mechanism of electron transfer in cytochrome c.......................................................... 4 4.1. Role of architecture in eukaryotic supercomplexes. . . . . . . . . . . . . . . ........................................................ 4 4.2. Cytochrome cbinding sites in complexes III and IV . . . . . . . . . . . . . . ........................................................ 6 4.3. Role of physical contacts and long-range electron tunneling. . . . . . . ........................................................ 6 5. Concluding remarks . . . . . . . . . . . . . . . . ..................................................................................... 8 Declaration of Competing Interest . . . . ..................................................................................... 8 Acknowledgements. . . . . . . . . . . . ......................................................................................... 8 References . . . . ........................................................................................................ 8 1. Introduction In biology, major vectorial energy transduction processes—such as photosynthesis and oxidative phosphorylation (OxPhos)—take place by electron transfer (ET) coupled to proton translocation https://doi.org/10.1016/j.ccr.2021.214233 0010-8545/Ó2021 Elsevier B.V. All rights reserved. ⇑ Corresponding author. E-mail addresses: [email protected] (I. Díaz-Moreno), [email protected] (M.A. De la Rosa). Coordination Chemistry Reviews 450 (2022) 214233 Contents lists available at ScienceDirect Coordination Chemistry Reviews journal homepage: www.elsevier.com/locate/ccr its two partners, whereas the alternative carrier, Cyt c 6 , binds loosely to the two membrane complexes [135–139]. This results in a dynamic ensemble of conformations rather than a single, well-defined complex. Notably, Pc has a small (type I) mononuclear blue-copper site, whereas Cyt c 6 shows structural features very close to those of respiratory Cyt c, including a large cofactor (such as a heme group) that supports multiple ET pathways. Thus, Pc might donate or accept electrons in close contact with its partner, as inferred from X-ray absorption spectroscopy (XAS) and MD data showing conformational fine-tuning of the Pc redox site [140,141] through an evolutionarily conserved arginine residue capable of modulating the redox potential [142]. In contrast, Cyt c 6 hovers on its partner surface, such that multiple paths across solvent could be available to mediate ET. Electrochemical tunneling measurements performed with the PSI complex reveal a behavior similar to that of Cyt c 1 , highlighting the possible role of electrostatically stabilized water molecules in long-range ET [143]. In agreement with these observations, the recently reported structure of mammalian Cyt c-CIV complex contains three water molecule layers in the long intermolecular space between the proteins, and a small contact surface [112]. Having long-distance ET between suitably orientated redox protein partners could yield a productive complex while avoiding the requirement of establishing tight van der Waals interactions and a well-defined conformation between partners. This possibility would reconcile having weak binding yet also having high specificity, thereby maintaining a high turnover and fast ET rates in the crowded environment of living cells (Fig. 5). 5. Concluding remarks The reduction in the dimensionality of partner searching during the courtship of Cyt cwith CIII and CIV—and in particular, when shifting the dimensionality from 3D to 2D—speeds up the coupling process and, consequently, ET. Such a 3D-diffusion-to-2D-sliding transition is enhanced by the assembly of CIII-CIV into SCs, in which Fe-Cu A distance shortening allows the formation of a continuous negatively-charged platform for landing and taking-off of Cyt cmolecules. Indeed, exhaustive ET analyses with the soluble electron carrier Cyt crevealed that several binding sites for Cyt c–proximal and distal–are available at the negatively-charged route on the CIII and CIV surfaces. Taken altogether, these data reinforce the concept of a ‘‘restrained 2D sliding pathway” of Cyt cmolecules– in contrast to 3D diffusion in the IMS–in the context of the plasticity model for the mitochondrial respiratory chain, in which the CIII-CIV SC assembles in response to metabolic needs. Restrained 2D sliding of Cyt cmolecules might indeed be governed by dipolar pre-orientation of the positive lysine-rich patch surrounding the Cyt cheme cleft with respect to the negatively-charged surfaces of CIII and CIV. Such a dipole–dipole orientation is essential for defining the ET transfer pathway in a weak, transient intermediate state. Up to now, the central dogma of energy metabolism in biology establishes that ET involves physical contact between proteins, with a consequent coupling between their redox centers. In this article, however, we review recent examples of tunneling in which proteins are able to transfer electrons over long distances in aqueous solution, without the need for direct, close contact. It is indeed tempting to think that the increase in redox potential gap induced by physical approach–rather than by physical contact–between partners might contribute to reach an optimal balance between ET rate and turnover. This opens up new avenues of understanding the mechanisms of ET, in which a landscape of multiple and dynamic long-distance conformational ensembles compatible with ET would replace the classical view of a single, well-defined shortdistance orientation. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors wish to thank Sofia Pauleta for her kind invitation to contribute this review article to the Coordination Chemistry Reviews Special Issue in honor of Isabel and José Moura for their 70 th anniversary. MAR, in particular, wants to express his profound gratitude to Isabel and José for a long friendship and congratulations for their exceptional scientific contributions. The authors’ work herein reviewed was supported by research grants from the Ministry of Science and Innovation (PGC2018- 096049-B-I00, FEDER/Ministerio de Ciencia e Innovación – Agencia Estatal de Investigación), European Regional Development Fund (FEDER), Andalusian Government (BIO-198, US-1254317, US- 1257019, P18-FR-3487 and P18-HO-4091, US/JUNTA/FEDER, UE), University of Seville (VI PPIT) and the Ramón Areces Foundation. GP-M was awarded a PhD fellowship from the Spanish Ministry of Education, Culture and Sport (FPU17/04604). References [1] R.A. 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Gorostiza, Electrochemically gated longdistance charge transport in Photosystem I, Angew. Chem. – Int. Ed. 58 (2019) 13280–13284, https://doi.org/10.1002/anie.201904374. G. Pérez-Mejías, A. Díaz-Quintana, A. Guerra-Castellano et al. Coordination Chemistry Reviews 450 (2022) 214233 11 Review Articles 277 Review II: Post-translational modifications of cytochrome c in cell life and disease  Guerra-Castellano A, Márquez I, Pérez-Mejías G, Díaz-Quintana A, De la Rosa MA, Díaz-Moreno I. 2020. “Post-translational modifications of cytochrome c in cell life and disease”. Int. J. Mol. Sci., 21: 8483. International Journal of Molecular Sciences Review Post-Translational Modifications of Cytochrome cin Cell Life and Disease Alejandra Guerra-Castellano, Inmaculada Márquez , Gonzalo Pérez-Mejías , Antonio Díaz-Quintana , Miguel A. De la Rosa and Irene Díaz-Moreno * Instituto de Investigaciones Qu í micas (IIQ), Centro de Investigaciones Cient í ficas Isla de la Cartuja (cicCartuja), Universidad de Sevilla, Consejo Superior de Investigaciones Científicas (CSIC), Avda. Américo Vespucio 49, 41092 Sevilla, Spain; [email protected] (A.G.-C.); [email protected] (I.M.); [email protected] (G.P.-M.); [email protected] (A.D.-Q.); [email protected] (M.A.D.l.R.) *Correspondence: idiazmor[email protected] Received: 20 October 2020; Accepted: 7 November 2020; Published: 11 November 2020   Abstract: Mitochondria are the powerhouses of the cell, whilst their malfunction is related to several human pathologies, including neurodegenerative diseases, cardiovascular diseases, and various types of cancer. In mitochondrial metabolism, cytochrome cis a small soluble heme protein that acts as an essential redox carrier in the respiratory electron transport chain. However, cytochrome cis likewise an essential protein in the cytoplasm acting as an activator of programmed cell death. Such a dual role of cytochrome cin cell life and death is indeed fine-regulated by a wide variety of protein post-translational modifications. In this work, we show how these modifications can alter cytochrome cstructure and functionality, thus emerging as a control mechanism of cell metabolism but also as a key element in development and prevention of pathologies. Keywords: cytochrome c; mitochondrial diseases; post-translational modifications 1. Introduction Mitochondria are the powerhouses of the cell. Their dysfunction favors the development of neurodegenerative diseases, cardiovascular pathologies, and several types of cancer [ 1 ]. A key protein in mitochondrial metabolism and control of redox signaling is cytochrome c(Cc) [ 2 ]. Ccis a small soluble protein (ca. 12 kDa, 104 amino acids) with four α -helices and a heme group, which is covalently bound by two cysteine residues (Cys14 and Cys17). The heme group is wrapped in a hydrophobic crevice, being only slightly exposed to the solvent. This conformation allows Ccto efficiently exchange electrons with its redox partners. Several residues of Ccundergo post-translational modifications, which provide a fine-tuned regulation at the functional level [ 3 ]. In the absence of stimuli, Ccis located in the mitochondrial intermembrane space, participating in redox metabolism [ 4 ]. However, several stimuli—such as apoptotic or DNA damage signals—promote Ccrelease from mitochondria. The first extramitochondrial target reported for Ccwas Apoptosis protease-activating factor-1 (Apaf-1) [ 5 ]. The interaction of Ccwith Apaf-1 in the cytosol is an early event of the mitochondrial apoptotic pathway [ 3 ]. Actually, the mitochondrial and extramitochondrial network of Ccencompasses a variety of proteins that are located in different organelles and cytosol [ 3 , 6 , 7 ]. These interactions can modulate the cell fate decision between cell life and death [ 7 ], which in turn relate to health and disease. The objective of this review is to carry out an in-depth analysis of how Ccmodifications affect its functions and their impact on the development of certain diseases and pathologies. Int. J. Mol. Sci. 2020,21, 8483; doi:10.3390/ijms21228483 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020,21, 8483 2 of 20 2. The Pleiotropic Role of Cytochrome cin Cell Homeostasis and Diseases Ccis a moonlighting protein whose localization and functions depend on cellular conditions (Figure 1) [ 3 , 6 , 7 ]. Under homeostasis, it is a key component of cellular energy metabolism, acting as an electron carrier between the cytochrome bc 1 complex (complex III, CIII) and cytochrome coxidase (complex III, CIV) in the mitochondrial electron transport chain (ETC) [ 4 ]. In this context, two binding sites have been described in cytochrome c 1 (Cc 1 ; from CIII) and CIV for Cc: a proximal site, optimal for electron transfer, and a distal site, which is not productive in terms of electronic transfer. It has been postulated that the interaction at the distal site could have physiological relevance in the dynamics and organization of electronic flow. It also seems to increase the local concentration of Ccavailable near the proximal site, allowing a rapid turnover of Ccmolecules [ 8 , 9 ]. Recently, the molecular basis of electron transfer from Cc 1 in CIII to Cchas been described [ 10 , 11 ]. According to the data, the coupling of redox potential shifts the conformational cycle of the Rieske subunit and the binding of Ccto Cc 1 causes electrons to flow in a single direction [ 11 ]. Cchas other redox functions, acting as a reactive oxygen species (ROS) scavenger and participating in the import of redox-coupled cysteine-rich proteins via Erv1-Mia40 [ 12 – 14 ]. The heme protein is also localized in vacuoles and zymogen granules during cell homeostasis, although its role there is unknown [15]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 21 Cc is a moonlighting protein whose localization and functions depend on cellular conditions (Figure 1) [3,6,7]. Under homeostasis, it is a key component of cellular energy metabolism, acting as an electron carrier between the cytochrome bc1 complex (complex III, CIII) and cytochrome c oxidase (complex III, CIV) in the mitochondrial electron transport chain (ETC) [4]. In this context, two binding sites have been described in cytochrome c1 (Cc1; from CIII) and CIV for Cc: a proximal site, optimal for electron transfer, and a distal site, which is not productive in terms of electronic transfer. It has been postulated that the interaction at the distal site could have physiological relevance in the dynamics and organization of electronic flow. It also seems to increase the local concentration of Cc available near the proximal site, allowing a rapid turnover of Cc molecules [8,9]. Recently, the molecular basis of electron transfer from Cc1 in CIII to Cc has been described [10,11]. According to the data, the coupling of redox potential shifts the conformational cycle of the Rieske subunit and the binding of Cc to Cc1 causes electrons to flow in a single direction [11]. Cc has other redox functions, acting as a reactive oxygen species (ROS) scavenger and participating in the import of redox-coupled cysteinerich proteins via Erv1-Mia40 [12–14]. The heme protein is also localized in vacuoles and zymogen granules during cell homeostasis, although its role there is unknown [15]. Figure 1. Cell localization and functions of cytochrome c. Cc is located in the mitochondrial intermembrane space, vacuole and zymogen granules under homeostasis. However, during DNA damage and apoptosis stimuli, mitochondrial Cc travels into the nucleus, the rough endoplasmic reticulum (RER) and the cytoplasm, respectively. The most relevant functions performed by Cc in each location are explained in the boxes. Color key: blue, physiological functions; red, functions performed under stress; and black, unknown functions. Created with BioRender.com (https://biorender.com/). Under nitro-oxidative stress conditions, Cc contributes to ROS production via the p66 redox cycle and acts as an inducer of programmed cell death (PCD) (Figure 1) [16]. Alterations in PCD signaling are relevant in many diseases [17,18]. Recent data indicates that the apoptotic network involving Cc is complex, and several target proteins are functionally equivalent in humans, plants, and Drosophila melanogaster [19–25]. At the onset of apoptosis, a Cc population—tightly bound to the inner mitochondrial membrane—catalyzes peroxidation of phospholipids, particularly cardiolipin (CL) (Figure 1). The nature of the Cc/CL interaction is subject of intense research [26]. Some authors proposed that CL-adducted Cc undergoes a profound tertiary conformational rearrangement that opens an entry channel for H2O2 molecules, enhancing the peroxidase activity of Cc [5,27]. Extensive data in the literature hints that the interaction with the CL affects the dynamics and conformation of Figure 1. Cell localization and functions of cytochrome c. Ccis located in the mitochondrial intermembrane space, vacuole and zymogen granules under homeostasis. However, during DNA damage and apoptosis stimuli, mitochondrial Cctravels into the nucleus, the rough endoplasmic reticulum (RER) and the cytoplasm, respectively. The most relevant functions performed by Ccin each location are explained in the boxes. Color key: blue, physiological functions; red, functions performed under stress; and black, unknown functions. Created with BioRender.com (https://biorender.com/). Under nitro-oxidative stress conditions, Cccontributes to ROS production via the p66 redox cycle and acts as an inducer of programmed cell death (PCD) (Figure 1) [ 16 ]. Alterations in PCD signaling are relevant in many diseases [ 17 , 18 ]. Recent data indicates that the apoptotic network involving Ccis complex, and several target proteins are functionally equivalent in humans, plants, and Drosophila melanogaster [ 19 – 25 ]. At the onset of apoptosis, a Ccpopulation—tightly bound to the inner mitochondrial membrane—catalyzes peroxidation of phospholipids, particularly cardiolipin (CL) (Figure 1). The nature of the Cc/CL interaction is subject of intense research [ 26 ]. Some authors proposed that CL-adducted Ccundergoes a profound tertiary conformational rearrangement that opens an entry channel for H 2 O 2 molecules, enhancing the peroxidase activity of Cc[ 5 , 27 ]. Extensive data Int. J. Mol. Sci. 2020,21, 8483 3 of 20 in the literature hints that the interaction with the CL affects the dynamics and conformation of a set of loops that determine the heme environment and iron coordination of Cc. In particular, the bond between the S δ atom of Met80 and iron can be disrupted, leading to high spin penta-coordinated species resembling that described for myoglobin [ 28 ]. It has been proposed that Cc/CL conjugates are sufficient for the formation of mitochondrial pores, which allow the release of the hemeprotein into the cytosol during apoptosis and redistribution of CL from the inner to the outer mitochondrial membrane [ 29 , 30 ]. In the early stages of apoptosis, Ccinteracts with the inositol 1,4,5-trisphosphate (IP3) receptor in the rough endoplasmic reticulum (RER) and with the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane. These interactions, in turn, promote calcium output and input, respectively (Figure 1) [ 31 – 33 ]. This traffic of calcium ions is restricted in space since both the IP3 receptors and the VDAC are located in mitochondria-associated endoplasmic reticulum membranes (MAMs) [ 34 , 35 ]. MAMs are microdomains involving both RER and mitochondrial proteins. These interconnection zones have fundamental roles in the transmission of calcium ions but are closely related to cellular lipid and energy metabolism [ 36 , 37 ]. The interaction of Ccwith the IP3 receptor prevents its calcium-induced inhibition, triggering the release of calcium [ 38 ]. Calcium binds to VDAC dimers, resulting in oligomerization [ 39 ]. This event increases the conductance of the channel and allows the release of Ccthrough it [ 40 – 42 ]. It is worth mentioning that the release of Ccinto the cytosol may not always lead to cell death, and the heme protein can sometimes act as a cell differentiation inductor (Figure 1) [ 20 , 43 ]. Ccalso reaches the cell nucleus upon DNA damage where it sequesters histone chaperones and inhibits chromatin remodeling (Figure 1) [ 23 , 24 ]. The pleiotropy of the effects of extramitochondrial Cccan be modulated by the cytosolic diflavin reductase NDOR1, which has NADPH-dependent Ccreductase activity [44]. 3. Post-Translational Modifications of Cytochrome c: Regulation, Functionality, and Structural Changes Post-translational modifications (PTMs) in proteins are regulatory mechanisms which control an ample set of cell metabolic processes and provide a tool to increase the functional diversity of proteins [ 45 – 47 ]. Indeed, PTMs play an integral role in regulating Ccfunctions [ 48 , 49 ]. The heme protein can undergo different PTMs (see below) that affect its physicochemical properties, interactions with physiological partners and, consequently, its functions. However, these effects depend on which residues are post-translationally modified. 3.1. Phosphorylation The functionality of Ccis controlled in vivo by phosphorylation of the following residues: Thr28, Ser47, Tyr48, Thr58, and Tyr97 [ 50 – 55 ]. Since Thr58 is replaced by isoleucine in humans, this position will not be a subject of this review. The low yield of phosphorylated Ccduring purification procedures makes functional and structural analysis challenging. Furthermore, the specific kinases responsible for phosphorylating the protein remain unknown—although some authors suggest that it could be the AMP kinase [ 53 ]. Therefore, it is common to mimic targeted phosphorylation with site-directed mutations that encode canonical or non-canonical (AMBER-type stop codons) substitutions. The evolved tRNA synthetase technique allows the site-specific incorporation of a non-canonical amino acid into the protein sequence [ 56 ]. The method is based on the use of an orthogonal aminoacyl-tRNA synthetases/tRNA pair. The aminoacyl-tRNA synthetase identifies the non-canonical amino acid and charges it in the tRNA. This is a special tRNA that recognizes AMBER-type stop codons, allowing the introduction of the residue into the desired position. The evolved tRNA synthetase technique can be used for the incorporation of non-canonical amino acids into proteins being produced in both prokaryotic and eukaryotic cells [57–59]. The use of non-canonical amino acids aims at a better emulating the physical properties of the target residues—e.g., the volume and/or charge—as compared to canonical substitutions (Table S1) [ 60 – 77 ]. A good example is the non-natural residue p-carboxymethyl-L-phenylalanine (pCMF), which has been Int. J. Mol. Sci. 2020,21, 8483 10 of 20 5. Clinical Relevance of Post-Translationally Modified Cytochrome c Proper working of Ccis essential for cell homeostasis and detoxification. Consequently, its malfunction is related to several mitochondrial diseases and aging [ 159 , 164 , 165 ]. The cause–effect relationships between post-translationally modified Ccand several pathologies have been widely studied. Figure 3graphically summarizes all the described modifications of Ccthat are present in human pathologies. Carbonylation, along with glycation, promote protein aggregation—this includes Cc, which is one of the principal features in neurodegenerative diseases [ 99 , 146 ]. On the contrary, Ccphosphorylated in positions Ser47 and Tyr97, or nitrosylated has been revealed as a neuroprotective agent against these neuronal disorders [ 49 , 55 , 81 , 123 ]. A plausible explanation is that these modifications inhibit PCD and enhance electron transfer under oxygen deprivation [ 81 ]. These findings, along with the fact that non-canonical amino acid-based phosphorylated Ccmutants are resistant to phosphatases, lead us to propose them as neuroprotectors with promising therapeutic applications. Cchomocysteinilation occurs in cardiovascular diseases [ 113 ] and, together with phosphorylation, in tumors [ 166 ]. Cancer cells are characterized by a high growth rate, which derives from enhanced mitochondrial metabolism, as well as an evasion of PCD (Table 1) [ 131 , 167 ]. Bazylianska and co-workers identified the acetylation of Lys53 in Ccin prostate cancer. In fact, they showed that acetylation strongly inhibits the role of Ccin apoptosis, allowing the survival of cancer cells [ 95 ]. Interestingly, tyrosine nitration and phosphorylation are mutually exclusive modification events. Thus, the Tyr-nitrated Ccspecies could function as antagonists to the Tyr-phosphorylated species, acting as an anticancer agent [ 123 ]. Moreover, proteins nitrated by peroxynitrite ion (including Cc) trigger inflammatory processes [ 168 ]. Even though Ccsulfoxidation has been identified in patients with thrombocytopenia, there is no evidence that this PTM directly causes the disease [169]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 21 undergo PTMs are labelled. Right, the different foldon units are colored as follows: foldon I in blue, foldon II in green, foldon III in yellow, foldon IV in red, and foldon V in purple. 5. Clinical Relevance of Post-Translationally Modified Cytochrome c Proper working of Cc is essential for cell homeostasis and detoxification. Consequently, its malfunction is related to several mitochondrial diseases and aging [159,164,165]. The cause–effect relationships between post-translationally modified Cc and several pathologies have been widely studied. Figure 3 graphically summarizes all the described modifications of Cc that are present in human pathologies. Carbonylation, along with glycation, promote protein aggregation―this includes Cc, which is one of the principal features in neurodegenerative diseases [99,146]. On the contrary, Cc phosphorylated in positions Ser47 and Tyr97, or nitrosylated has been revealed as a neuroprotective agent against these neuronal disorders [49,55,81,123]. A plausible explanation is that these modifications inhibit PCD and enhance electron transfer under oxygen deprivation [81]. These findings, along with the fact that non-canonical amino acid-based phosphorylated Cc mutants are resistant to phosphatases, lead us to propose them as neuroprotectors with promising therapeutic applications. Cc homocysteinilation occurs in cardiovascular diseases [113] and, together with phosphorylation, in tumors [166]. Cancer cells are characterized by a high growth rate, which derives from enhanced mitochondrial metabolism, as well as an evasion of PCD (Table 1) [131,167]. Bazylianska and co-workers identified the acetylation of Lys53 in Cc in prostate cancer. In fact, they showed that acetylation strongly inhibits the role of Cc in apoptosis, allowing the survival of cancer cells [95]. Interestingly, tyrosine nitration and phosphorylation are mutually exclusive modification events. Thus, the Tyr-nitrated Cc species could function as antagonists to the Tyr-phosphorylated species, acting as an anticancer agent [123]. Moreover, proteins nitrated by peroxynitrite ion (including Cc) trigger inflammatory processes [168]. Even though Cc sulfoxidation has been identified in patients with thrombocytopenia, there is no evidence that this PTM directly causes the disease [169]. Figure 3. Cause–effect of post-translational modifications of cytochrome cin human pathologies. Carbonylation, glycation and nitration of specific residues of Ccpromote the development of several neurodegenerative diseases and strokes. However, phosphorylated Ccat positions 47 and 97, or nitrosylated in its tyrosine residues has been revealed to be a neuroprotective agent against this type of pathology. Cancer cells show many modifications of heme protein, such as phosphorylations, acetylations, homocysteinilations, and nitrations. Nitrosylation and phosphorylation are mutually exclusive modifications, so the presence of nitrosylated Ccspecies could be act as an anticancer agent. Int. J. Mol. Sci. 2020,21, 8483 11 of 20 Notably, homocysteinilation is also related to cardiovascular diseases. Finally, it has been described that peroxynitrite ion and nitrated proteins (including Cc) trigger inflammatory processes. The green wheels represent Ccmodifications that favor the development of diseases (see the direction of rotation of the gear indicated by the green arrows), white wheels represent the pathologies—gray arrows represent the direction of rotation that produces the development of the disease—and the red wedges indicate PTMs that impede them. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/22/ 8483/s1. Author Contributions: Conceptualization, A.G.-C., I.M., G.P.-M., A.D.-Q., M.A.D.l.R. and I.D.-M.; resources, A.G.-C., I.M. and G.P.-M.; writing—original draft preparation, A.G.-C., I.M. and G.P.-M.; writing—review and editing, M.A.D.l.R. and I.D.-M.; supervision, M.A.D.l.R. and I.D.-M.; funding acquisition, M.A.D.l.R. and I.D.-M. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Ministry of Science and Innovation [PGC2018-096049-B-I00), European Regional Development Fund [FEDER), Andalusian Government [BIO-198, US-1254317, US-1257019 and P18-FR-3487), VI PPIT-US and TA Instruments. G.P.-M. were awarded a PhD fellowship from the Spanish Ministry of Education, Culture and Sport [FPU17/04604). Acknowledgments: Figure 1were created using BioRender icons [https://biorender.com/). Evolutionary analyses were performed with the ConSurf Server [https://consurf.tau.ac.il/). Conflicts of Interest: The authors declare no conflict of interest. Abbreviations AcK Apaf-1 N-ε-acetyl-L-Lys Apoptosis protease-activating factor-1 CL Cardiolipin CIII Complex III CIV Complex IV Cytochrome cCc Cytochrome c1Cc1 CcO Cytochrome coxidase ECT Electron transport chain IP3 Inositol 1,4,5-trisphosphate MAMs Mitochondria-associated endoplasmic reticulum membranes PCD Programmed cell death pCMF pI p-carboxymethyl-L-phenylalanine Isoelectric point PTMs Post-translational modifications RER Rugose endoplasmic reticulum RNS Reactive nitrogen species ROS Reactive oxygen species VDAC Voltage-dependent anion channel References 1. Pieczenik, S.R.; Neustadt, J. Mitochondrial dysfunction and molecular pathways of disease. Exp. Mol. Pathol. 2007,83, 84–92. [CrossRef] [PubMed] 2. D í az-Moreno, I.; Garc í a-Heredia, J.M.; D í az-Quintana, A.; De La Rosa, M. Á . Cytochrome c signalosome in mitochondria. Eur. Biophys. J. 2011,40, 1301–1315. [CrossRef] [PubMed] 3. 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Berezin, C.; Glaser, F.; Rosenberg, J.; Paz, I.; Pupko, T.; Fariselli, P.; Casadio, R.; Ben-Tal, N. ConSeq: The identification of functionally and structurally important residues in protein sequences. Bioinformatics 2004,20, 1322–1324. [CrossRef] two of them providing axial ligands for the heme iron. His18 at the proximal side of the heme provides the imidazole ligand conserved among the class I family. At physiological pH values, Met80 thioether acts as a distal ligand. The heme porphyrin ring is covalently bound to the protein backbone by thioether bonds between the vinyl groups of the porphyrin and conserved cysteine residues in the CXXCH motif. For human Cc, conserved cysteine residues are Cys14 and Cys17. According to hydrogen exchange (HX) experiments, the apparently simple structure hides five folding units (called foldons) with different stabilities [79]. The most stable one (I) comprises the N- and C-terminal α-helices. Foldon II comprises Ω I (from Thr19 to Phe36) and α-helix 3, which comes before Ω III . Foldon III (a.k.a. neck) comprises two short amino acid stretches with an extended conformation flanking the Ω II -loop. Notably, the latter faces heme propionates and is the least stable foldon (V), followed by the Ω III region (IV) containing Met80. The low stability of the loop containing Met80, comprising the sixth iron ligand, has a crucial role in Ccphysiology. Recent ultrafast X-ray spectroscopy analyses have highlighted the weakness of the Met80-S δ -Fe +2 bond and the lack of stability (4kJmol -1 ) provided by the protein matrix, most likely via hydrogen bonding [80]. At physiological pH, Cchas a net charge of +8 from its unevenly distributed ionizable groups [81, 82]. This favors interactions with negatively charged molecules, such as the polar head of phospholipids, including CL. This interaction was first analyzed by Kimelberg and Lee, using lecithin-CL vesicles [83]. Their analysis together with early HX measurements by solid-state nuclear magnetic resonance (ssNMR) indicated that Ccpreferentially binds with CL [84]. They also suggested that during this interaction, CL destabilizes or unfolds the hemeprotein. Further relaxation time measurements by 31 P NMR showed that Ccimpacts CL dynamics [85]. Surface plasmon resonance and electrochemical experiments on planar lipid bilayers allowed Salamon and Tollin to propose a two-step mechanism [86–88]. According to their proposal, Ccfirst binds to membranes through electrostatic interactions and, subsequently, through hydrophobic interactions to promote changes in both the structure of Cc and the membrane. Then, electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) analyses showed that Ccundergoes structural changes which affect Fe coordination and result in the appearance of a radical at high liposome concentrations [89]. Hence, mixing Ccwith lipids may yield several species, found in recent fluorescence anisotropy analyses [90]. Apparently at odds with this proposed model, paramagnetic-quenching EPR experiments on horse heart Cc, spin-labelled at different lysine positions, indicated that the hemeprotein can weakly interact with 1,2-dioleoyl-sn- glycero-3-phosphoglycerol (DOPG) bilayers [91]. This study highlighted three lysine residues at Ω III (K72, K86, and K87, a.k.a A-site; Figure 2(a)) adjacent to the DOPG membrane. Further fluorescence studies using vesicles containing fluorescent lipid probes identified a secondary CL-binding site at low pH values [93]. Contrary to the A-site, CL association at this novel region (a.k.a. C-site; Figure 2(a)) is unaffected by ionic strength or the presence of ATP. Data suggested CL binds to this site via hydrogen bonds at N52, and a single acyl chain of the phospholipid inserts into a nearby hydrophobic pocket while the others remain in the bilayer [94]. This proposed interaction mechanism is known as the extended lipid anchorage model and is supported by studies on the ability of natural and engineered phospholipids to quench the fluorescence of Zn-substituted Cc[95, 96]. Consistently, a N52I mutation heavily impacts the kinetics of the interaction between Ccand CL in CL-containing liposomes [97]. While, a combination of lysine modification, tryptic digestion, and MALDI-TOF analysis unveiled that horse heart Ccpromotes the fusion of lipid vesicles via an interaction at a second positive patch. This region (L-site) comprises K22, K25, K27, H26, and H33, besides the previously reported A- and C-sites (Figure 2(a)) [98]. An additional UV-Vis analysis showed slight differences in the binding kinetics within a set of yeast Ccmutants [99]. Based on their own data and the solution structure of L-site N-site A-site C-site H18 M80 (a) K72 K73 K79 K55 K8 K7 K22 K100 K25 K27 R38 K39 K53 ⁎ ⁎ (b) I81 I75 A51 F10 V3 V11 A101 L32 A43 F46 S15 V20 F36 M80 (c) Figure 2: Cardiolipin-binding sites in cytochrome c. (a) Ribbon representation of oxidized human Cc(PDB 2N9J) [92]. CL-binding sites are highlighted in orange (A-site or distal productive), green (L-site or proximal productive), purple (C-site), and cyan (N-site or proximal unproductive). The heme axial ligands H18 and M80 are highlighted as well. (b) Side chain representation of the positively charged Cc residues involved in the formation of the Cc-CL complex. Residues marked with an asterisk are reported to constitute the L- and N-sites. (c) Side chain representation of hydrophobic Ccresidues, which ensure the tight interaction between Ccand CL acyl chains. 4 Oxidative Medicine and Cellular Longevity the protein (PDB 1AKK; [100]), the authors proposed a cleft defined by the Ω III residues K72, K73, K86, and R91, matching A-site. Building on from this data, the ability of site-directed mutants of horse heart Ccto bind CL- containing liposomes was tested [101]. Notably, substituting K73 and K79 with asparagine alters the affinity of Cctowards these liposomes, whereas the same mutation at position 72 does not. A common feature of these binding sites is the presence of several positively charged and hydrophobic residues (Figures 2(b) and 2(c)). In line with Tollin and Salamon’s early postulates [88], the interaction between the positively charged residues of Ccand negatively charged phosphate groups of CL initiates the formation of the Cc-CL complex driven by electrostatic interactions [101–104]. After the initiation of the complex, hydrophobic Ccresidues play a key role in the interaction with CL acyl chains, thereby establishing a tight binding between Ccand CL [15, 105]. A strong piece of evidence supporting the extended anchorage hypothesis is the presence of a channel formed by residues 52 to 74 in the tuna CcXRD structure [81], where the highlighted cavities are only visible in the 4Å model. An attempt to manually dock CL inside the structure of horse heart Ccyielded two acyl chains within the backbone [97]. Any assessment of clashes of these two acyl chains with internal residues was missing. Nevertheless, none of the two cavities in this structure appear in the updated structure at 1.5Å resolution (PDB 5CYT; unpublished). Hence, the extended lipid anchorage model demands that Ccmust undergo a substantial conformation change when interacting with lipid vesicles. A low-resolution analysis using monoclonal antibodies suggested that lipid-bound Ccdisplayed an alkalinelike conformation [106]. The alkaline form of oxidized Cc presents a conformation different to that of the native species, which relates to an exchange between the Met thioether ligand and a Lys amine. However, to our knowledge, the only Ccalkaline structure available (PDB 1LMS; [107]) lacks a channel in which the acyl chain may be lodged, although the heme moiety is more accessible to solvent than the native structure. Another possibility is the formation of Ccoligomers by domain swapping [108, 109]. Although these structures are highly variable depending on how the domain swapping is triggered by experimental conditions, a recent analysis has shown they are capable of encompassing an acyl chain [110]. Furthermore, Tyr67 in these structures would face C 11 of docked linoleic (18:2 Δ9,12 ) acid, a finding in agreement with peroxidation mechanisms. However, evidence of Ccoligomerization in the presence of membranes remains unavailable. Many of the studies above report the loss of the Met80 coordination and at least partial denaturation—or transition to a molten globule state—of Ccwhen binding to CL- containing vesicles or liposomes [15, 84, 93, 95–97, 109– 111]. Aside from CL, some other lipids can elicit similar such structural changes in Cc[105]. This makes the heme moiety more accessible to small substrates such as carbon monoxide or nitric oxide [105, 111]. Moreover, time-resolved fluorescence resonance energy transfer (trFRET) experiments show labelled residues—not previously reported to bind CL—moving further away from the heme group in the presence of CL-containing liposomes [103, 112]. Furthermore, changes in the CD spectra and Trp59 fluorescence indicate unfolding of at least the lowest energy foldons. Consistent with the loss of Met80 coordination, substantial negative shifts in the midpoint potential of Ccare observable in the presence of lipid vesicles [113, 114]. Disruption of the Fe-Met80 bond also correlates with a substantial increase in Ccperoxidase activity when Ccinteracts with CL-containing vesicles [15, 105, 113]. An illustration of conformational changes, adapted from Muenzner et al. [112], is available in Figure 3(a). The current understanding of the effects of lipid binding on Ccstructure (as described above) is controversial. For instance, depending on the nature of the CL-rich membrane preparations and experimental setup, the shift in redox potential can be positive [86] or negative [113, 114]. Recently, Wand and collaborators demonstrated that the mitochondrial crypts are concave surfaces, opposed to the convex lipoic vesicles often used in the analyses. They then analyzed the solution structure of oxidized horse heart Ccencapsulated in reverse lipid micelles by NMR [115]. They utilized pseudocontact shifts as experimental restraints, which are highly sensitive to distances and orientation with respect to the main axes of the iron coordination sphere. An overlay of the structures of free [100] and encapsulated horse Cc [115] (PDB 1AKK [100] and 2N3B [115], respectively) are shown in Figure 3(b). Notably, the structure of Ccremains unaltered, and the chemical-shift perturbation map [115] resembles that observed for interactions between Ccand its protein targets [116] and other class I single-heme cytochrome cfamily members [117]. In fact, this patch includes a novel region (N-site), not previously reported, comprising F36, G37, T58, W59, and K60, besides the known residues of the A- and L-sites (Figure 2(a)). Surprisingly, ssNMR spectra tracking the interaction between Ccand small unilamellar (convex) vesicles showed no feature indicating formation of either a molten globule or unfolding [118]. Consistently, solution HSQC (NMR) spectra of horse heart Ccin the presence and absence of similar vesicles overlapped quite well [119]. Although some signals broaden, while others were displaced—as expected for this interaction—no features of unfolding are apparent. Therefore, the overall curvature or the membrane may be irrelevant for the stability of bound Cc. Previous data on Cc unfolding in the presence of lipid vesicles might be reviewed in regard to membrane composition, lipid stability, and experimental setup. For instance, the dynamics of Cccan change depending on buffer conditions [119]. Similarly, the ionization state of CL also affects Ccbinding [120]. Understanding the entire landscape of the data requires the rationale underpinning the studies to be taken into account. An equilibrium between unbounded native Ccspecies and membrane-bound populations—by either weak electrostatic interactions, hydrogen bonding, or hydrophobic interactions—can be observed. The relative weights of such populations vary with experimental conditions. Furthermore, our ability to detect different species relies on the sensitivity of each biophysical approach. As recently pointed out, 5Oxidative Medicine and Cellular Longevity many of the analyses above suffer from low-resolution data or the introduction of probes that could partially alter results [118]. Therefore, full understanding requires complete knowledge of experimental conditions. 4. Interplay between Lipid and Cytochrome c Dynamics: The Compact/Extended Model Cccan undergo several structural transitions triggered by changes in experimental conditions such as pH [89, 90, 121–123]. For example, low-spin (S=0)Fe II species may turn into high-spin (S=2) species in the presence of liposomes, as shown by EPR and MCD spectroscopies [89]. Notably, ionic strength and lipid-to-protein (L/P) ratio strongly influence the populations of the distinct species [15, 110, 124, 125]. These ratios relate to lipid surface coverage by Ccmolecules [125, 126]. At low L/P ratios the Cccoated micelles undergo coalescence—forming giant unilamellar vesicles—and precipitate. Whereas at moderate L/P ratios, Ccpromotes interactions between small unilamellar vesicles [125, 127]. The dynamic of the Ccheme group is highly sensitive to spin state, axial ligand strength, and conformational changes. Therefore, Raman spectroscopy studies have been key in unveiling the complexity of Ccconformation equilibria under different conditions [123, 125–128]. Hildebrandt and collaborators detected native (B1; His-Met coordination) and altered (B2) states in the presence of DOPG vesicles [125]. The B2 state comprises different species: a low-spin (B2[6cLS]), His-His-coordinated species, and two high-spin I II II III V H18 M80 A-site IV L-site N-site C-site Changes in protein structure (a) V I II II A-site IV III V L-site N-site A-site N-site II IV III II I L-site Ω–Loop Helix-I C-site C-site H18 M80 H18 M80 Changes in protein dynamics (b) Figure 3: Proposed model for the interaction of cytochrome cwith cardiolipin at pH values above 7. (a) Upper: structure of free Ccshowing the foldon units (in red scale, PDB 1AKK [100]). Lower: the Cc-CL interaction promotes unfolding of the metalloprotein and dissociation of the axial ligand M80, thus increasing accessibility to the heme crevice. (b) Upper: structural comparison of free (in red scale, PDB 1AKK [100]) and CL-bound Cc(in blue scale, PDB 2N3B [115]). Lower: interaction of Ccwith CL yields a slight difference in dynamics at the level of the Ω-loops and helix-I. The different foldon units of Ccare colored as a gradient from the most stable (dark colors) to the weakest region (light colors). The heme group is in green, and the iron atom in orange. 6 Oxidative Medicine and Cellular Longevity species (a pentacoordinated (B2[5cHS]) and a hexacoordinated species (B2[6cHS])) in which a water molecule acts as the sixth ligand. The populations of the different states change according to the L/P ratio. The B2 species predominates at high L/P ratios, whereas native B1 and His-His- coordinated B2[6cLS] coexist at lower ratios. These states are also detectable by MCD [114]. The B2 bis-His- coordinated species is detectable when Ccis absorbed onto self-assembled monolayers, with and without CL [129]. At increasing concentrations of DOPC/tetraoleoylcardiolipin (TOCL) micelles, the population of bis-His species increases, as confirmed by His-by-Asn mutations and spectroscopic analyses [130]. In addition to the L/P ratio, the content of CL and its composition influence Ccaffinity and dynamics in the bound state. Fluorescence data has indeed revealed that increasing amounts of CL favors Ccbinding to membranes [120]. The theoretical analysis therein suggests that the protonation state of CL may have a strong influence on populations of distinct membrane-bound Ccspecies. However, the authors acknowledge that the formalism does not include the effects that Ccexerts on CL distribution or membrane state (see below). Indeed, kinetic investigations have shown that the exchange rate between a native-like, compact (C) and the “extended”(E) conformations correlates with the amount of CL in the vesicles [131]. A more recent spectroscopic analysis on titration experiments by Pandiscia and Schweitzer-Stenner resulted in similar conclusions [90]. Additionally, the study highlighted that the L/P ratio also affects the relative weight of electrostatic interactions, hydrogen bonds, and hydrophobic interactions—according to ionic strength series. In summary, besides governing Ccconformational states, the L/P ratio modulates the nature of bilayerprotein interactions. Notably, all the studies highlighted above hint at a rather peripheral binding model, with little or no embedding of Ccinto the membrane [90, 123, 125, 126, 128, 129, 131]. Ontheotherhand,Ccdoesexertastronginfluenceonlipid head-group dynamics, as revealed by early 31 P ssNMR studies whichdemonstratedanincrease inacylchainsdynamicsanda restraint in the polar head groups of phospholipids [132, 133]. Interestingly, Cchas little impact on the 31 P ssNMR “powder” spectra of dioleoyl-phosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), or DOPC/DOPE vesicles not containing CL [134]. Indeed, NMR data strongly supported CL undergoing phase separation—to form CL rafts—within mixed DOPC/CL preparations upon the addition of Cc[132, 135]. Further, freeze-fracture electron microscopy images highlighted the ability of Ccto promote the transition of phospholipid bilayers containing CL into non-bilayer structures, including inverted tubular (H II ) states [133, 134]. A full isotropic signal in the 31 P ssNMR spectra of phospholipid preparations in the presence of Ccevinced the formation of vesicular or micellar structures when the vesicles contained CL [132, 136]. Similarly, a downfield broad signal indicated that CL mediates the formation of the H II phase upon the addition of Cc. In this sense, molecular dynamics simulations in which Ccis in contact with a DOPC/CL membrane highlight the ability of this protein to recruit CL into rafts [118]. In addition, Cccan induce local changes in membrane curvature whenthe ratio of CL increases up to 20%. Furthermore, Ccinduces pore formation in DOPC/CL giant unilamellar vesicles (GUV), as shown by confocal microscopy [137]. These pores are wide enough to allow Ccand dextran molecules to cross the membrane. Nevertheless, the ability of Ccto induce membrane changes seems to be secondary regarding the activation of peroxidase activity. Addition of Ccto large DOPC/CL unilamellar vesicles at a ca. 6 CL/Ccratio promotes peroxidase activity without substantially affecting 31 P ssNMR spectra or the 13 C frequencies of the lipid glycerol signals [138]. The major population—those accounting for less than 10% of the protein are not detectable—of Ccin these experiments displays the same structure as the native protein in solution. Chemical-shift perturbation analysis revealed that residues affected include the Ω III -loop and some nearby residues (including A-site residues). However, changes in the dynamics of the Ω III -loop as it couples with bilayer motions are observable. This, rather than an overall unfolding, is sufficient to trigger the peroxidase activity under these conditions. In accordance with this finding, the perturbation pattern shifts when the temperature is changed or when the vesicle phospholipids are unsaturated. The formation of Cc-CL complex requires approximately 6 molecules of CL per Ccmolecule [15, 112, 119, 138, 139]. The values of the apparent dissociation equilibrium constant for the Cc-CL-reduced complex are in the low micromolar range (1.4μM at pH 8.1 and 2.2μMat pH 6.5) [140], whereas in the oxidized form, they are in the high micromolar range in a two-step reaction (20μM and 42μM) [102]. Nevertheless, binding constants depend on CL composition. The affinity of Cctowards tetrastearyl-cardiolipin-containing vesicles is several fold higher than that for TOCL ones [102], whereas tetra-myristoyl- cardiolipin barely interacts Cc[15]. Notably, the measured affinities correlate well with the peroxidase activity of Cc in the presence of the respective vesicles, rather than the degree of unsaturation in the acyl chains [102]. 5. Cardiolipin Oxidation by Cytochrome c CL is particularly sensitive to auto-oxidation processes—those directly initiated by inducers, such as ROS. The proximity of its four unsaturated acyl chains allow “arm-to-arm”propagation, enhancing its reactivity [141]. Auto-oxidation takes place in several steps. A free radical (e.g., a ROS molecule) contains an unpaired electron, and this semioccupied orbital is a sink for a second electron. Polyunsaturated fatty acids (PUFA), such as linoleic or linolenic composing CL, are particularly sensitive to ROS-induced oxidation due to conjugative effects. Radicals such as superoxide, peroxyl (ROO • ), or hydroxyl (HO • ) sequester a hydrogen atom from the α-meth- ylene carbon with respect to the first (di-) vinyl group. The resulting radical reacts immediately with O 2 to generate a peroxyl radical. The variation of electron vacancy in the lipid radical underlies the diversity of reaction products. No matter how the lipid radical originates, it tends to propagate via 7Oxidative Medicine and Cellular Longevity a reaction with molecular oxygen, water, or other lipids. Within the process of CL signaling, there are several reactions which stand out including the addition of oxygen (to form peroxides), transfer of hydrogen atoms, addition of peroxyl radicals, and intramolecular peroxide substitution [142]. Oxidative phosphorylation and certain mitochondrial enzymes are sources of O 2-• radicals [143]. Superoxide is highly soluble in lipids but can be reduced within membranes by tocopherol and quinone and eliminated by superoxide dismutase (SOD), which transforms two superoxide molecules into a molecular oxygen and hydrogen peroxide [144]. In fact, enzymes like SOD, catalase, and peroxidases take part in active cell defense against oxidative stress [144]. H 2 O 2 is a strong oxidant—the reduction potential for the H 2 O 2 /H 2 O pair is +1.35V, at pH 7—but kinetically inefficient. However, the presence of iron chelates under certain pathological conditions can enhance H 2 O 2 efficiency through Haber-Weiss-like reactions (see Equations (1)–(3)) [145]: Fe3+ +H 2O2⟶Fe2++O−∙ 2+2H +ð1Þ Fe2+ +H 2O2⟶Fe3+ +HO −+HO∙Fenton’s reaction  ð2Þ O−∙ 2+Fe 3+ ⟶Fe2+ +O 2ð3Þ The hydroxyl radical product is highly reactive, sequestering hydrogen atoms from available substrates. The resulting carbon-centered radicals may react with molecular oxygen to generate (hydro-) peroxides (Equations (4)–(6)): HO∙+RH⟶H2O+R ∙ð4Þ R∙+O 2⟶RO∙ 2ð5Þ RO∙ 2+H ++Fe 2+ ⟶ROOH + Fe3+ ð6Þ Nevertheless, homeostatic cells exert a tight control over metal chelation to avoid Fenton’s reactions. Indeed, a set of antioxidant agents prevent a surge in the levels of ROS. Hence, except for pathological conditions, H 2 O 2 requires the activity of peroxidases to function efficiently as an oxidant. Within the IMM, Ccdisplays both peroxidase and oxygenase activities, the latter promoting CL oxidation, while sparing other phospholipids [18, 146]. This event is crucial for the release of mitochondrial proapoptotic factors into the cytoplasm [18]. 5.1. Peroxidase and Oxygenase Activities of Cytochrome c (Dr. Jeckyll and Mr. Hyde). Heme peroxidases constitute a vital and ubiquitous group of heme enzymes catalyzing the twoelectron oxidation of substrates using H 2 O 2 as the ultimate electron acceptor [147, 148]. In canonical peroxidases, H 2 O 2 is added to the pentacoordinated, high-spin Fe III . The resulting state—Compound I—is two oxidation equivalents above the resting state and is reduced back to the resting configuration in two steps through Compound II. Both states, Compounds I and II, are high valence oxoferryl (Fe IV ) derivatives, but the first comprises an additional π-cation radical (see Equations (7)–(9)) (Figure 4) [147]. Heme FeIII  RS +H 2O2⟶Heme O = FeIV−R∙+  CompI +H 2O ð7Þ Heme O = FeIV−R∙+  CompI +AH 2⟶Heme O = FeIV  CompII +AH ∙ð8Þ Heme O = FeIV  CompII +AH 2⟶Heme FeIII  RS +AH ∙∙ +H 2O ð9Þ FeIV O FeIV O ROOH ROH AH AH HN HN HN N N N A Compound I Compound II O OH Tyr 48 Tyr 67 Tyr 74 OH OO C5H11 C5H11 C5H11 O2 + FeIII H2O + A Figure 4: Peroxidase and oxygenase activities of cytochrome c. Reaction model merging the proposal from Kagan and collaborators [149] and the adapted catalytic model of cyclooxygenases as reviewed by Marnett [150]. Blue arrows correspond to the canonical peroxidase cycle [146]. Heterolytic cleavage of a peroxide substrate—preferentially for Cc, a lipid hydroperoxide—yields the corresponding hydroxyl derivative (or water when the substrate is H 2 O 2 ) and Compound I, which is reduced back to the resting ferric state in two sequential singleelectron transfers from A substrate. Red and green arrows indicate the reactions purportedly leading to oxygenase activity according to the literature. Spin trap experiments have detected Y48 radicals [151]. Dimers of tyrosines 67 and 74 and oxidation products of Y48 are detectable even in the absence of H 2 O 2 [152]. The tyrosyl radical sequesters a hydrogen atom from an unsaturated fatty acid. Finally, O 2 reacts with the alkyl radical to form an alkyl peroxide radical as an initial product undergoing further reactions. 8 Oxidative Medicine and Cellular Longevity In true peroxidases, a histidine residue acts as an acidbase catalyst at the distal side of the heme ring, while a highly conserved arginine residue stabilizes the alkoholate leaving group to favor the heterolytic cleavage of the peroxide O–O bond [153]. The orientation of these residues and the hydrogen-bond network at the heme distal side are critical for efficient formation of Compound I [154]. As recently pointed out by Vlasova [148], the composition of a true peroxidase active site prevents its damage by highly oxidizing intermediate compounds. Ccand other hemeproteins can act as pseudoperoxidases; that is, under only certain stimuli they show peroxidase activity [148, 155]. Contrary to true peroxidases, the surroundings of heme moiety are unprotected against oxidation, so the peroxidase activity ends up damaging the protein. In the early 1990s, Radi and collaborators reported the ability of Ccto oxidize small compounds in solution [145] and to carry out lipid peroxidation [146] in the presence of H 2 O 2 . Nevertheless, the reactivity of Cctowards H 2 O 2 was low, as it requires the absence of the sixth ligand. Thus, the Kmvalue for H 2 O 2 was very high—ca. 65mM. In this sense, oxidative reactions showed a time lag after the addition of H 2 O 2 , indicative of an activation step. Moreover, peroxidase activity in solution decayed at pH values higher than 8 [145]. Poor reactivity may also result from the absence of histidine at the distal site (vide infra), as it is an acid-base residue “pumping”the heterolytic cleavage of H 2 O 2 during Compound I formation. Based on chemiluminescence analyses of the reaction, Chance and coworkers proposed a homolytic reaction mechanism (Equations (10)–(12)) [156], supported by EPR spin trap studies of small organic hydroperoxides [157]. Heme FeIII  +H 2O2⟶Heme HO∙ −FeIII  ð10Þ HO∙+H 2O2⟶H2O+HO ∙ 2ð11Þ Heme HO∙ −FeIII  +HO ∙ 2⟶Heme FeIII  +O 2+H 2O ð12Þ However, EPR spin trap experiments highlighted the generation of Cctyrosine radicals upon treatment with H 2 O 2 [18, 151, 155, 158]. Furthermore, spin trap experiments detecting radical products resulting from oxidation of different substrates by H 2 O 2 strongly suggested the reaction being mediated by an oxoferryl [O=Fe IV ] intermediate [155]. Analysis of the orientation of this radical within the native state identified a tyrosine residue at the Ω II -loop—namely, Y48 in horse heart Ccand either Y46 or Y48 in human Cc[159]. The peroxidase activity of Ccin the presence of CL increases by three orders of magnitude when the driving oxidant is a lipid peroxide instead of H 2 O 2 [149]. Spin trap analysis of reaction products by Kagan and collaborators indicates a diversity of catalytic mechanisms depending on the binding site of the substrate, namely, a homolytic peroxide cleavage minority mechanism and a major, heterolytic mechanism. Notably, the small hydroperoxide substrates involved in this pathway dock near R38 and H33. However, how the docked structure undergoes conformational changes to fulfil all geometrical constraints needed for Compound I formation remains unclear (Figure 4). Binding of hydrogen peroxide to the heme iron is a key step in the reaction mechanism underlying peroxidase activity. The reactive species need to displace the thioether axial ligand. This takes place when a strong interaction between Ccand a membrane induce a substantial conformational change in the hemeprotein [15, 84, 93, 95–97, 109, 111]. Nevertheless, Kagan and coworkers also detected peroxidase activity in Ccat low CL/Ccratios—at which the most interactions are weak electrostatic [15]. Furthermore, they found that the energy required to activate peroxidase activity is lower than that required for partial unfolding of the protein. In fact, as pointed out before, the bond joining iron to the thioether ligand is quite weak [160]. Thus, “breathing”fluctuations of Ω II - and Ω III -loops may facilitate the replacement of the thioether ligand by small reactants—such as cyanide, carbon monoxide, water, or hydrogen peroxide—without demanding major structural changes. In fact, Ccperoxidase activity rises in the presence of H 2 O 2 as the concentration of denaturant increases, as previously observed in a similar analysis with bacterial cytochrome c 550 [161]. Statistical analysis of activity and unfolding slopes indicate that increasing the motions of the weakest Ω-loops correlates well with peroxidase activity in the “compact”Ccspecies [162]. The peroxidase activity of Cccan exert a protective role in mitochondria under certain conditions [11]. Indeed, reduction of lipoid hydroperoxide compounds to hydroxyl ones provides a way of relieving oxidative stress in the mitochondrial membrane while generating signaling molecules [149]. Moreover, O 2-• reduces nitric oxide ( • NO) generated in mitochondria under stress to form peroxynitrite (HOONO), a highly reactive species. Cc-CL complexes have been proposed to aid peroxynitrite detoxification to yield either nitrate or nitrite through an oxoferryl state [163]. Conversely, the peroxidase activity of Cchas been implicated in certain pathologies. For instance, oxidation of sulfite to its radical SO 3-• is a key mechanism in sulfite toxicity [164]. Moreover, Ccmediates the formation of tyrosine radicals responsible for α-synuclein dimerization [66, 165], which leads to the development of the Lewy body diseases. Canonical peroxidase activity involves two sequential one-electron oxidation steps and no transfer of oxygen from the oxoferryl complexes to the substrate [146]. Nevertheless, Compound I in certain heme enzymes—such as cytochrome P450—can transfer oxygen to certain substrates yielding hydroxy-derivatives. Interestingly, hydroxy-derivatives of CL cannot undergo peroxidation and inhibit the release of Cc[166]. Altogether, considering the findings above concerning tyrosyl radicals in Cc[151], Kagan and collaborators proposed that Ccacts as an oxygenase to produce CL peroxidation [167]. This activity would also be responsible for phosphatidylserine peroxidation affecting the plasma membrane during apoptosis [65]. This hypothesis suggests that hydrogen is transferred to Compound I from a nearby tyrosine residue to yield oxoferryl Compound II and the aforementioned tyrosyl radical (Figure 4). This mechanism is similar to that proposed for cyclooxygenases, in which a hydrogen atom is sequestered from an acyl chain, generating 9Oxidative Medicine and Cellular Longevity a carbon-centered radical capable of reacting with molecular oxygen [150]. Unlike true peroxidases, the environment of the heme moiety is unprotected from highly oxidizing species arising during the catalytic cycle [148]. When reacting with H 2 O 2 , degradation of the heme porphyrin often becomes apparent by a diminution of the Soret band intensity [146, 156]. A thorough mass spectrometry analysis of Ccresidue adducts derived from H 2 O 2 has been carried out by Flemmig and collaborators [152]. Several oxidation reactions can occur to produce a methyl-sulfoxide derivative from the methionine thioether, a sulfonic acid derivative from cysteines and 2-oxohistidine from histidine. While tyrosine residues can covalently cross-link or undergo oxidation to dihydroxyphenylalanine (DOPA) and subsequently to quinones, lysine residues can undergo carbonylation [152, 168, 169]. These changes occur when H 2 O 2 is added to Ccsamples [80, 152, 170–173]. Remarkably, different regions in the protein display different sensitivities to oxidation depending on the environment. For instance, specific M80 oxidation takes place in the presence of DOPC/DOPE micelles [174]. The Ω III -loop is the first to be affected, whereas foldon I (helices I and IV) is the least affected by oxidation. Notably, the peroxidase activity of Ccincreases in a time-dependent manner upon the addition of H 2 O 2 . Such increments in peroxidase activity may result from successive oxidation of M80 and lysine residues, as proposed by Yin and Konermann [80, 170]. Indeed, M80 oxidation promotes conformation exchange in Cc which impacts on heme ligation. With time, oxidative damage extends to the porphyrin ring, releasing iron capable of performing Fenton’s reactions [173]. Finally, it is worth noting that CL peroxides can induce at least some of these oxidative PTM [152]. 5.2. The Alkaline Transition of Cytochrome c and Peroxidase Activity. As mentioned above, previous data obtained using monoclonal antibodies highlighted that an alkaline-like conformation could interact with CL and exit mitochondria during apoptosis [106]. These antibodies also recognize the M80A mutant in the cell nucleus [175]. Notably, this mutant displays enhanced peroxidase activity. In addition, the peroxidase activity of Ccis somewhat pH dependent [145, 176]. Indeed, for horse heart Cc, peroxidase activity increases at acidic pH values [177] and slows beyond pH 8 [145]. Furthermore, the ability of Ccto oxidize O 2-• falls at pH values above 7 [7]. The affinity of Cctowards membranes and the interaction patch involved also depend on pH [15, 98, 120, 127]. These effects illustrate how pH- dependent conformation changes modulate the different activities performed by Cc. A number of mutations and PTM have been reported to simultaneously affect the peroxidase activity of Ccwhile bringing the so-called alkaline transition to lower even physiological pH values [178–182]. Loss of M80 coordination is evident from NMR spectra and UV-Vis spectra in all these studies. Ccperoxidase activity requires the heme iron to be pentacoordinated; the relationship with the pKAof the alkaline transition could be attributable to the lysine amine being weaker than methionine thioether in the ligand. However, at a neutral pH, lysine is a stronger ligand than methionine [183]. In fact, horse heart and human Ccshow lower peroxidase activities at alkaline pH values [145, 182]. Moreover, mutation M100K in P.versutus cytochrome c 550 makes the protein more stable at neutral pH while decreasing its peroxidase activity 20-fold [183]. Nevertheless, the shift in the alkaline transition towards lower pH values indicates destabilization or higher dynamics in the Ω II - and Ω III -loops in the Met-coordinated species. Given the weakness of the thioether ligand bond towards iron, increasing fluctuations of these loops will increase the population of high-spin species and/or alternative low-spin (e.g., bis-His) species below the pKAvalue of the transition. This is observable in phosphomimic mutants, as well as in nitrated species of Cc[178–180, 184–186]. Furthermore, enhanced dynamics facilitate the access of small substrates to the heme iron [162]. 5.3. Control of Cardiolipin Oxidation by Post-translational Modification of Cytochrome c. Protein PTM regulate tightly controlled cellular processes and increase the functional diversity of proteins, often acting as a cell response switch. Several post-translational modifications modulate Ccstructure and functionality, such as sulfoxidation [187], carbonylation [152], homocysteinylation [188], nitration [179, 180], and phosphorylation [189] (Figure 5). Phosphorylation of tyrosine residues is associated with many human pathologies including cancer, ischemia, asthma, and sepsis. As highlighted earlier, tyrosine radicals are key for the oxygenase activity of Cc[157]. Thus, the amount of hydroxyl products from TOCL oxidation is lower when Y48E phosphomimic species instead of WT Ccacts as a catalyst [190]. Additionally, tyrosine phosphorylation impairs the formation of radicals, preventing dimerization [191]. This fact may be critical in pathological processes such as Parkinson’s disease [66]. Therefore, the PTM that affect these residues are key in regulating Ccactivity. Given the difficulty in preserving the phosphorylation state of Ccoutside of cell extracts, a common strategy to investigate consequences of phosphorylation is to mimic the modification by site-directed mutagenesis. All phosphomimetic Ccspecies, except a mutant at position 97, display altered affinity towards cardiolipin [178, 186, 190, 192–195]. The peroxidase activity of both free Ccand Cc-CL complexes increases in the phosphomimetic T28D and Y48pCMF. However, for the Y48E species, the increase only occurs with the free protein (Table 1) [178, 190, 192, 194]. In addition, at a high CL/lipid ratio, the peroxidase activity of the T28E mutant decreases [195]. A possible explanation may be that the greater population of CL versus other lipids in the liposome composition promote unfolding of the hemeprotein, acting as an offswitch (Table 1) [131]. Hence, the negative charge at these positions could induce structural changes in the heme crevice which allow greater accessibility for hydrogen peroxide (Figure 5). Peroxynitrite generated during nitrooxidative stress is a powerful amino acid modifier, affecting tyrosine residues among others [201]. Common products of the reaction 10 Oxidative Medicine and Cellular Longevity between tyrosine and HOONO are 3,5-dinitrotyrosine, 3- nitrotyrosine, tyrosine radicals, and dityrosine. Nevertheless, treatment of Ccin vitro with peroxynitrite yields its 3-nitrotyrosine adducts, with the nitro group attaching to one of the C ε of the aromatic ring [202]. Nitration affects the redox potential of Ccas well as its electron-exchange kinetics, depending on the residue involved [203]. The nitration of Y46, Y48, Y74, and Y97 residues also increases the peroxidase activity of Ccand lowers the pKAvalue of the alkaline transition besides other functional properties [179, 180, 183, 185, 197, 204, 205] (Table 1). Nitration of Cchas been associated with several diseases, including chronic nephropathy [206]. All modifications/mutations of S47 and Y67 alter the peroxidase activity of Cc[79, 179, 185, 192, 195, 197]. Y67 is located close to Met80 and is part of the hydrophobic pocket which houses the acyl chains of CL. This residue is also key for the stability of the ΩIII −loop (Table 1). Homocysteinylation is a PTM that involves the covalent bonding of a homocysteine thiolactone—an intermediate metabolite of methionine metabolism—with a lysine residue [188]. Upregulation of this PTM is implicated in several human pathologies including cancer and cardiopathies [198, 207]. The degree of homocysteinylation, as well as the rate at which this modification occurs in the presence of homocysteine thiolactone, is related to the number of lysine residues [188]. Human Ccdisplays a lysine content of 17.1% in its amino acid composition, which makes it sensitive to homocysteinylation. Homocysteinylation of surface lysines on Cccauses aggregation of the protein. However, N-homocysteinylated lysines adjacent to the heme cavity produce conformational changes, disrupt the coordination of the M80 axial ligand, and alter the redox state of Cc, reducing the iron of the heme group [196, 208]. These conformational changes increase Ccperoxidase activity (Table 1) [209]. As discussed before, Ccis modulated by several oxidative modifications due to its activity, eventually leading to changes in iron coordination. One oxidative modification is the carbonylation of lysine residues, which affects residues 72 and 73, both of which are involved in the alkaline transition of Cc[170] (Figure 5). Reportedly, successive carbonylation events at K53, K55, K72, and K73 lead to the formation of the pentacoordinated Ccspecies, resulting in increased Ccperoxidase activity (Table 1) [170]. Similarly, the sulfoxidation of M80 facilitates the formation of a Compound I-type intermediate that initiates the activity of Cc peroxidase (Table 1) [171, 210]. 5.4. Peroxidase Activity of Cytochrome c and Diseases. Since the peroxidase activity of Ccrelates to the activation of apoptosis, it is a clear target for the development of more efficient OO HO P OH HO O OHO HO N O O Nitration Phosphorylation Nitrotyrosine S O HO O O HO O Sulfoxidation Carbonylation Carbonylated lysine Methionine sulfoxide O OH H2N H2N H2N OH O OH NH2 NH2 NH2 NH2 OH OO H NH2 NH N O OH Glycine Serine Tyrosine Histidine Point mutations Phosphotyrosine NH 2 HN O HO O HS N-Homocysteinylation N-Homocysteinylated lysine Y74 Y67 Y46 M80 Y48⁎ S47 T28 K72 K73 K55 K8 K100 K53 K99 K13 K87 K86 Y97 G41 ⁎ Figure 5: Functional implications of PTM and mutations of cytochrome c. Left: chemical modifications of cytochrome cresidues. Right: ribbon representation of oxidized human Cc(PDB 2N9J) [92]. Residues are colored by type of PTM: pink for nitration, purple for sulfoxidation, orange for phosphorylation, brown for carbonylation, cyan for N-homocysteinylation, and blue for point mutations. Y48 (asterisk) can be either phosphorylated, nitrated, or mutated for histidine, while Y97 (asterisk) can be phosphorylated or nitrated. 11Oxidative Medicine and Cellular Longevity therapies against certain diseases or pathologies. There are several examples in the literature that shed light on this topic. The point mutations Y48H and G41S in Cccause the appearance of a special type of thrombocytopenia (thrombocytopenia 4) which is an asymptomatic disorder [199, 200]. Thrombocytopenia 4 is notable for normal platelet production. However, in thrombocytopenia platelets are not transported to the bone marrow and remain in the matrix, causing the effective platelet content to be lower. Both mutations cause an increase in peroxidase activity, which is related to an increase in the population of Ccin the pentacoordinated heme state [181, 211, 212] (Table 1). Most neurodegenerative diseases are associated with cellular stress and apoptotic processes. Due to the double role played by Ccin the electron transport chain and apoptosis, it represents an amenable target for the development of therapies against neurodegenerative pathologies. For example, post-translational phosphorylation of Ccat Y97 is an excellent neuroprotective strategy following brain injury as it increases the efficiency of electron transport during hypoxic conditions [193, 213]. Moreover, Cchas been implicated in the development of Parkinson’s disease—as it colocalizes with α-synuclein in the Lewy bodies [214]. In fact, the peroxidase activity of Ccplays an important role in the aggregation of α-synuclein by tyrosine dimerization [66, 174]. Minocycline is an antibiotic functional against both Gram-positive and Gram-negative bacteria. Additionally, it displays neuroprotective properties [215]. The antibiotic minocycline impairs the interaction between Ccand CL, inhibiting the activation of peroxidase activity and the consequent release of Ccinto the cytosol to trigger apoptosis [216–218]. Nitric oxide is a well-known inhibitor of Ccperoxidase activity and thus may downregulate apoptosis [219, 220]. Flavonoids are excellent antioxidants that prevent cellular aging by ROS scavenger activity. In addition, they can inhibit Ccperoxidase activity, preventing proapoptotic events [221]. The protection of healthy cells during radiotherapy is a hot topic. In fact, novel synthetic compounds—e.g., imidazole-substituted fatty acids—are currently being trialed in order to preserve healthy cells by inhibiting the peroxidase activity of Ccduring the irradiation process [222, 223]. These de novo compounds, mainly imidazole conjugates, block access to the heme crevice preventing the activation of peroxidase activity. Finally, the activation of proapoptotic events, such as the release of Ccafter the peroxidation of CL, may be a good target for the development of more efficient and specific therapies against cancer [224, 225]. 6. Concluding Remarks In this review, we have outlined major advances and hypotheses regarding the oxidation of CL by Cc. CL oxidation is a crucial event at the onset of a diverse range of pathologies, and thus, controlling it has become a key objective of current research. Targeting Cc—a key player in CL oxidation—has emerged as an important task. Since its discovery last century, Cchas displayed great functional complexity despite its apparently simple structure. Indeed, its highly dynamic architecture, which enables conformation changes critical in regulating metabolism, signaling, and cell fate, still amazes the scientific community. Ccinteracts with membranes in different ways depending on their composition and curvature, being able of modifying the latter. When the hemeprotein interacts with lipids, it may undergo subtle changes in the dynamics of its most flexible foldons or may even unfold. A plethora of factors, including PTM, control these phenomena. The chemical activity of this protein ranges from the simplest reactions to diverse and complex reaction mechanisms to drive the oxidation of substrates including CL. In the recent years, we have witnessed concerted effort to unveil the intimate chemistry of this process. Solving this Table 1: Effect of PTM and point mutations on cytochrome c peroxidase activity. CcPTMs/mutation Effect on peroxidase activity References Sulfoxidation a M80 ↑[168, 187, 196] Nitration b Y46 ↑[184] Y48 ↑[184] Y67 ↑[185, 197] Y74 ↑[185, 197] Y97 ↑[185, 197] Carbonylation c K53 ↑[152] K55 ↑[152] K72 ↑[152] K73 ↑[152] Phosphorylation T28 T28D ↑[192] T28 T28E ↓[195] S47 S47D ≈[192] Y48 Y48E ↑/↓[176, 190] Y48pCMF ↑[194] Y97 Y97E ≈[176] Y97pCMF ≈[193] N-Homocysteinylation d K8/K13 ↑[198] K86/87 ↑[198] K99 ↑[198] K100 ↑[198] Point mutation G41S ↑[181, 199] Y48H ↑[181, 200] a Determined under oxidative stress. b Determined after peroxynitrite treatment. c Determined after chloramine-T treatment. d Determined after homocysteine thiolactone treatment. 12 Oxidative Medicine and Cellular Longevity conundrum will require us to discriminate minority conformations in functional assays and elucidate how this activity is tuned under distinct conditions. 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