Effect of Electrostatic Immobilization on the Electrochemistry of Human and Horse Cytochrome c

E ec o elec os a ic immobiliza ion on he elec ochemis y o human and ho se cy och ome c Jos´ e Luis Olloqui-Sa iego a,* , I. M´ a quez a , Alejand a Gue a-Cas ellano b , M. Mole o a , Miguel A. De la Rosa b , Juan Jos´ e Cal en e a , I ene Díaz-Mo eno b , Ra ael And eu a,* a Depa amen o de Química Física, Uni e sidad de Se illa, P o eso Ga cía Gonz´ alez, 1, 41012, Se illa, Spain b Ins i u o de In es igaciones Químicas, cicCa uja, Uni e sidad de Se illa – Consejo Supe io de In es igaciones Cien í icas (CSIC), Am´ e ico Vespucio 49, 41092, Se illa, Spain ARTICLE INFO Keywo ds: Cy och ome c Immobiliza ion s eng h He e ogeneous elec on ans e Elec on ans e kine ics A henius pa ame e s Ma yusho ’s heo y ABSTRACT P o ein ilm ol amme y is a sensi i e ool o cha ac e ize he elec on ans e p ope ies o edox p o eins in a a ie y o en i onmen s and con o ma ional s a es. He e, a de ailed ol amme ic s udy aimed o explo e he e ec o elec os a ic immobiliza ion on he elec on ans e he modynamics and kine ics o adso bed human- and ho se- cy och ome c was ca ied ou . Fo his pu pose, he wo cy och omes we e adso bed on hiol monolaye s (SAM) wi h di e en immobiliza ion s eng hs and dono –accep o dis ances. While he modynamic edox pa ame e s do no seem o be a ec ed by he monolaye hickness and cha ge densi y, elec on ans e kine ics a e signi ican ly modula ed by he p o ein immobiliza ion s eng h. S onge p o ein–SAM elec os a ic in e ac ions esul in lowe elec on ans e a es in bo h non-adiaba ic and ic ion kine ic egimes. This beha io is u he cha ac e ized by smalle p e-exponen ial ac o s and ac i a ion en halpies in A henius ype plo s. These kine ic esul s in he physiologically ele an non-adiaba ic elec on ans e egime a e shown o be consis en wi h he ecen ly de eloped Ma yusho ’s heo e ical o mula ion o p o ein elec on ans e . Mo e- o e , a compa ison be ween he kine ic pa ame e s o he wo cy och ome a ian s suppo s he hypo hesis ha di e ences be ween hei elec on ans e a es o igina e in hei s uc u al lexibili y o accommoda e he con o ma ional changes equi ed o o m he p ecu so complex be ween cy och ome and a nega i ely cha ged edox pa ne . 1. In oduc ion Cy och ome c (Cc) is an elec on ans e ing p o ein, which is mainly ound in he in e memb ane egion o mi ochond ia. I is an essen ial componen in a la ge a ie y o me abolic p ocesses [1–3]. Unde homeos a ic condi ions, his p o ein beha es as an elec on shu le om cy och ome bc 1 complex o cy och ome c oxidase complex. Addi ionally, Cc has a numbe o al e na i e p oapop o ic unc ions such as a cell dea h induce analogous o a ca diolipin oxygenase, ac i a o o he caspase-dependen apop o ic ou e in he ea lies e en s o apop osis, as well as o he signaling pa hways. [4–11] This unc ional e sa ili y o Cc is in ima ely ela ed o i s high s uc u al lexibili y, which modula es i s edox p ope ies in di e en biological scena ios [12–17]. In ac , i has been epo ed ha he Cc en i onmen , including molecula c owding, pH and empe a u e [18–21], is c ucial o i s unc ional ac i i y and s abili y. Besides, an ex ensi e li e a u e de o ed o he cha ac e iza ion o he edox ac i i y o Cc e eals ha i s unc- ionali y is modula ed by i s in e ac ion wi h mi ochond ial pa ne s [22–28]. Pa icula ly, elec os a ic in e ac ions in p o ein–biomolecule binding e en s a e essen ial o con olling he edox unc ion o he p o ein, hough he de ailed molecula mechanism o his con ol is no ully unde s ood. Wi hin his con ex , in es iga ing he elec on ans e be ween immobilized Cc and elec ode su aces, ha mimic some as- pec s o he p o ein complex en i onmen du ing i s physiological unc ion, is expec ed o p o ide aluable in o ma ion on he in luence o p o ein docking condi ions on he modynamic and kine ic elec on ans e pa ame e s. F om he pionee ing s udy o Eddowes and Hill on he elec ochemical beha io o Ho se hea e icy och ome c a a gold elec ode modi ied wi h 4,4 ′ -bipy idyl [29], many wo ks ha e add essed om a undamen al poin o iew he in es iga ion o elec on ans e o a a ie y o mammalian Cc immobilized on o unc ionalized elec- odes [25,30–42]. Howe e , in sha p con as wi h he la ge numbe o * Co esponding au ho s. E-mail add esses: [email p o ec ed] (J.L. Olloqui-Sa iego), [email p o ec ed] (R. And eu). Con en s lis s a ailable a ScienceDi ec Jou nal o Elec oanaly ical Chemis y jou nal homepage: www.else ie .com/loca e/jelechem h ps://doi.o g/10.1016/j.jelechem.2025.118975 Recei ed 10 Decembe 2024; Recei ed in e ised o m 20 Janua y 2025; Accep ed 24 Janua y 2025 Jou nal o Elec oanaly ical Chemis y 981 (2025) 118975 A ailable online 30 Janua y 2025 1572-6657/© 2025 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license ( h p://c ea i ecommons.o g/licenses/by- nc-nd/4.0/ ). epo s dealing wi h he elec ochemical p ope ies o comme cially a ailable ho se- and bo ine-Cc, only a ew pape s ha e been de o ed o he s udy o he in e acial elec on ans e o human-Cc [23,42–44]. A compa ison o ho se- and human-Cc e eals ha 90 % o he amino acid sequence is conse ed and, speci ically, he sol en accessibili y and elec os a ic cha ge dis ibu ion a ound he ac i e si e a e nea ly iden- ical, so ha only small di e ences a e o be expec ed in ela ion o hei edox unc ionali y (see Fig. 1). Despi e hei s uc u al simila i ies, a ecen wo k has epo ed sig- ni ican di e ences in he dissocia ion equilib ium cons an o hei espec i e Cc: Cy och ome c oxidase (CcO) complexes [46]. In addi ion, Rod íguez–Rold´ an e al. [47] showed ha he complex be ween human- Cc and CcO exhibi s a ema kably lowe in acomplex elec on ans e a e han he analogous complex o med by ho se-Cc and CcO. Since bo h cy och omes display simila kine ics when hey a e educed by a se ies o la in semiquinones, hese au ho s sugges ed ha he obse ed di e ences in elec on ans e a e we e o igina ed in hei in e ac ions wi h hei na u al edox pa ne s o o m he p ecu so elec on ans e complex. Likewise, Wege ich e al. epo ed a as e sel –exchange elec on a e o ho se-Cc ha hey a ibu ed o a mo e adequa e p o- ein–p o ein in e ac ion in he icini y o he heme c e ice [48]. In addi ion, a highe alue o he in e acial elec on ans e a e cons an was ob ained again o ho se-Cc as compa ed o he human a ian (40 s −1 s. 4 s −1 a 25 ◦C) when hey we e elec os a ically immobilized on o sil e elec odes coa ed wi h an 11–me cap oundecanoic acid mono- laye [44]. Conside ing hese p e ious esul s, we in end o ca y ou in his wo k a de ailed elec ochemical compa ison o bo h cy och omes, by de e mining hei he modynamic and kine ic elec on ans e pa- ame e s when hey a e adso bed on o nega i ely cha ged hiol sel - –assembled monolaye s (SAMs) o di e en leng hs and cha ge densi ies. The in e acial elec on ans e a e cons an s we e ini ially analyzed in e ms o he ansi ion be ween non-adiaba ic and ic ional kine ic egimes as he elec ode-p o ein dis ance is dec eased. Then, a u he dissec ion in o p e-exponen ial and ac i a ion en halpy e ms o each kine ic limi was pe o med by conside ing he empe a u e dependence o he wo limi ing a e cons an s. Fo he wo cy och omes, ansi ion om s ong o weak immobiliza ion s eng h is accompanied by an inc ease o bo h p e-exponen ial and ac i a ion en halpy alues. In he non-adiaba ic egime, his esul ag ees wi h he expec ed con i- bu ions o p o ein-elec ode dis ance oscilla ions o he elec on ans e a e cons an in Ma yusho ’s model [49]. Quali a i ely simila esul s we e ob ained in he ic ional egime, in b oad ag eemen wi h he expec ed highe ac i a ion ene gies and slowe molecula mo ions in he p esence o a s ong immobiliza ion ield. Compa ison be ween kine ic pa ame e alues o he wo p o eins shows ha di e ences end o disappea as he immobiliza ion s eng h dec eases, he e o e sugges - ing ha hey o igina e in he p o ein-monolaye in e ac ion. 2. Expe imen al sec ion 2.1. P o eins and chemicals Comme cial ho se hea Cc was pu chased om Sigma Ald ich. Esche ichia coli (E. coli) BL21 (DE3) cells we e ans o med wi h pBTR1- WT plasmid o ecombinan ly exp ess human Cy c. P o ein exp ession and pu i ica ion was ca ied ou as p e iously desc ibed [50]. B ie ly, cells we e cul u ed a 30 ◦C and 150 pm o 20 h in LB medium sup- plemen ed wi h 100 μ g_mL −1 ampicillin. Cells we e ha es ed by cen i uga ion (10 min a 9 000 g) and suspended in lysis bu e (10 mM T icine-NaOH, pH 8.5, 0.02 mg mL −1 DNase, 1 mM phenyl- me hylsul onyl luo ide and comple e p o ease inhibi o ). The esus- pended cells we e physically up u ed by sonica ion and we e hen cen i uga ed (30 min a 14 000 g and 4 ◦C) o cellula deb is disca d. The supe na an was loaded in a Nu ia-S (Bio-Rad, He cules, CA, USA) column o p o ein pu i ica ion using a FPLC sys em (Bio-Rad). The pu i y o p o ein samples was es ed by UV–Vis spec opho ome y in a Jasco® V-650 spec opho ome e . The A 280 /A 550 a io o he esul ing Cc p epa a ions in he educed s a e was abou 1.1, as p e iously e- po ed [50]. P o ein concen a ion was measu ed by isible spec o- pho ome y, using a alue o 28.92 mM −1 cm −1 o he ex inc ion coe icien o he educed species a 550 nm. 3–me cap op opionic acid (MPA), 8–me cap ooc anoic acid (MOA), 11–me cap oundecanoic (MUA), acid, 3–me cap op opanol (MPOL), 8–me cap ooc anol (MOOL) and 11–me cap oundecanol (MUOL) we e om Sigma Ald ich, and we e used wi hou u he pu i ica ion. Bu e solu ions we e p epa ed om anhyd ous sodium dihyd ogen phospha e, di–sodium hyd ogen phospha e, bo h pu chased om Fluka, and Milli- po e wa e . 2.2. Elec ochemical measu emen s Linea cyclic ol ammog ams we e eco ded wi h an AUTOLAB PGSTAT-30, om Eco Chemie B.V, in a h ee-elec ode undi ided glass cell. The cell was equipped wi h a P ba coun e elec ode, an Ag/AgCl/ NaCl sa u a ed e e ence elec ode and a gas low sys em. The e e ence elec ode was connec ed o he cell solu ion ia a sal b idge and kep a oom empe a u e (298 ±2 K) in a non-iso he mal con igu a ion. Re- po ed po en ial alues we e co ec ed o he no mal hyd ogen elec ode (NHE) po en ial scale by adding +192 mV o he expe imen al alues. The wo king elec ode was a polyc ys alline gold disc wi h a 0.0314 cm 2 geome ic a ea. All measu emen s we e ca ied ou unde a gon a mo- sphe e. Wo king solu ions we e 20 mM sodium phospha e bu e solu- ions a pH 7.0. P io o measu emen s, he gold su ace was successi ely polished wi h 0.3 and 0.05 µm alumina and insed wi h Millipo e wa e . Then, he elec ode was sonica ed in e hanol o 5 min o emo e any esidual alumina, d ied wi h pu e ni ogen, and chemically cleaned wi h Fig. 1. Ribbon ep esen a ion o ho se-Cc (blue, PDBid 1AAK) [45] and human-Cc ( ed, PDBid 2N9J [17]). Va ia ions in amino acid composi ion a e highligh ed. J.L. Olloqui-Sa iego e al. Jou nal o Elec oanaly ical Chemis y 981 (2025) 118975 2 a “pi anha” solu ion. Then, he gold su ace was unc ionalized by imme sing he elec ode in o an e hanolic solu ion o ei he 1 mM ω -me cap oca boxylic acid o o a mix u e o 1 mM ω -me cap o- ca boxylic acid plus 2.5 mM ω –hyd oxy–n–alkane hiol, o 1 h a 277 K. Then, p o ein immobiliza ion was ca ied ou o 90 min a 277 K, by deposi ion o a 15 µL d op o a 12 µM Cc and 10 mM sodium phospha e bu e pH 7.0 solu ion on o he modi ied elec ode. The modynamic and kine ic aspec s o he elec on exchange be ween p o ein and elec ode we e assessed om he a ia ion o he ol amme ic esponse wi h empe a u e and po en ial scan a e, espec i ely. The measu ing em- pe a u e ange was se be ween 0 ◦C and an uppe empe a u e limi ha was dic a ed by p o ein he mal deso p ion. Cyclic ol ammog ams we e eco ded a scan a es be ween 0.02 and 200 V s −1 , and posi i e eedback o ohmic d op compensa ion was applied whene e he po- en ial scan a e was highe han 1 V s −1 . Though bo h double laye capaci ance (C dl ) and solu ion esis ance (R s ) a y wi h expe imen al condi ions, such as na u e o he hiol monolaye o empe a u e, ypical alues o C dl ( o he sho e hiol monolaye s) a e ~1 μ F and o he uncompensa ed solu ion esis ance R us ~ 100 Ω, so ha he ime con- s an τ =R us C dl o cha ge he double laye wi h posi i e eedback compensa ion is ~0.1 ms. 3. Resul s and Discussion 3.1. Elec on ans e he modynamics o immobilized Cc Tempe a u e– a iable cyclic ol amme y was employed o de e - mine he en opic and en halpic changes accompanying he elec on exchange o human-Cc and ho se-Cc wi h he elec ode. Addi ionally, we ha e also assessed he e ec o he elec os a ic immobiliza ion s eng h on he edox beha io o bo h p o eins, by compa ing hei ol amme ic esponse when hey a e adso bed on ei he a highly cha ged monolaye o ω -me cap oca boxylic acid, o on a weakly cha ged monolaye , which includes a mix u e o a ω -me cap oca boxylic acid and a ω –hyd oxy–n–alkane hiol. This ype o immobiliza ion b ings in o play he elec os a ic in e ac ions ha Cc expe iences when i comes in o con ac wi h some o i s physiological pa ne s, such as Cy och ome c 1 , CcO, o he anionic phospholipid ca diolipin [26,27]. Fig. 2 illus a es some ypical ol ammog ams eco ded a low scan a e o human- and ho se-Cc. They a e associa ed wi h he heme Fe 3+ / Fe 2+ edox con e sion and display a gaussian shape, ypical o su ace- con ined edox species. We didn’ obse e any addi ional ol amme ic peaks a mo e nega i e po en ials (see Fig. S1), which sugges s he absence o p o eins wi h non-na i e con o ma ions. A low scan a es, ol ammog ams a e cha ac e ized by a small sepa a ion o hei ca hodic and anodic peak po en ials (~8 mV) and by ull wid hs a hal heigh o ~92 mV (FWMH) a 25 ◦C, close o he heo e ical alue (90.6 mV) o a monoelec onic ans e o a popula ion o iden ical and non- in e ac ing edox cen e s. By in eg a ing he ol amme ic peaks, we ob ained p o ein co e ages o ~10 ±4 pmol cm −2 , i. e. somewha below a p o ein monolaye [51]. While he FWMH alues emain almos cons an wi hin he en i e ange o empe a u es, he amoun o elec- oac i e p o ein dec eases upon inc easing empe a u e, as expec ed o a he mally induced p o ein deso p ion. The midpoin po en ial alues (E 1/2 ) o immobilized human- and ho se-Cc a 25 ◦C and pH 7 (see Table 1) we e signi ican ly lowe han he alue o ~260 mV s. NHE de e mined o bo h p o eins in solu ion [42,52], which is consis en wi h a ela i e s abiliza ion o he p o ein e ic o m ollowing i s adso p ion on a nega i ely cha ged hiol monolaye , and i has been obse ed be o e o o he mammalian, yeas , and bac e ial Cc [25,52–55]. Mo eo e , i espec i e o he alkane chain Fig. 2. Cyclic Vol ammog ams o human-Cc ( ed and o ange lines) and ho se Cc (blue and cyan lines) immobilized on o a gold elec ode modi ied wi h mixed ( op panel) o pu e (bo om panel) hiol monolaye s, whose hyd oca bon chain-leng h inc eases om le o igh . The suppo ing elec oly e was a 0.02 M sodium phospha e bu e solu ion o pH 7, and he ol ammog ams we e eco ded a 0.5 V s −1 and 25 ◦C. Table 1 The modynamic pa ame e s o he Fe(III)/Fe(II) edox con e sion o immobi- lized human- and ho se-Cc. Monolaye Cc E1/2/ mV a ΔS0 c/ J K −1 mol −1 ΔH0 c/ kJ mol −1 MPA:MPOL Human 205 ±5–86 ±5–45 ±3 Ho se 208 ±5–68 ±5–40 ±3 MPA Human 195 ±5–84 ±5–44 ±3 Ho se 201 ±5–73 ±4–41 ±3 MOA:MOOL Human 187 ±5–94 ±7–46 ±6 Ho se 195 ±5–83 ±6–44 ±4 MOA Human 180 ±5–88 ±5–44 ±3 Ho se 194 ±5–74 ±5–41 ±3 MUA:MUOL Human 180 ±5–98 ±5−46 ±4 Ho se 185 ±5–96 ±5−46 ±5 MUA Human 175 ±5–101 ±6−47 ±4 Ho se 185 ±5–96 ±6−46 ±5 a Measu ed a 25 ◦C, pH 7. J.L. Olloqui-Sa iego e al. Jou nal o Elec oanaly ical Chemis y 981 (2025) 118975 3 leng h and composi ion o he hiol monolaye , he E 1/2 alues o human-Cc a e somewha lowe han hose o ho se-Cc, indica ing a highe sensi i i y o human-Cc owa ds i s elec os a ic in e ac ion wi h he monolaye . I is also in e es ing o no e in Table 1 a sys ema ic end owa ds mo e nega i e E 1/2 alues (by ~ 20 mV) as he molecula hickness o he SAM inc eases. The same obse a ion was made be o e by Mu gida e al. [22] o ho se-Cc immobilized on pu e me cap o- ca boxylic acid SAMs deposi ed on sil e elec odes, which hey in e - p e ed quan i a i ely in e ms o he elec os a ic model o Smi h and Whi e [56]. Howe e , he ac ha essen ially he same E 1/2 a ia ion (see Fig. S2 in he Suppo ing In o ma ion sec ion) is ob ained i e- spec i e o he na u e o he me al subs a e, o o he ionized cha ge densi y in he SAM, sugges s an al e na i e explana ion in e ms o in insic p ope ies o he monolaye s, such as a change in hei dipola con ibu ion o he su ace po en ial as he molecula chain-leng h o he hiol a ies. In his ega d, i should be no ed ha he po en ial o ze o cha ge o a MUA monolaye deposi ed on Au(1 1 1) was shown o be 50 mV mo e nega i e han ha o a MPA monolaye a pH 7 [57], and ha he obse ed E 1/2 a ia ion can easonably be ep oduced om he change o alkane hiol dipole momen s wi h molecula chain-leng h e- po ed p e iously [58], by assuming an e ec i e pe mi i i y alue o 13 inside he monolaye s (see he Supo ing In o ma ion). To ob ain he en opy (ΔS0 c) and en halpy (ΔH0 c) educ ion changes, he a ia ion o E 1/2 wi h empe a u e was de e mined in a non- iso he mal cell con igu a ion, so ha [59–61]: ΔS0 c =nF( ∂ E1/2 ∂ T)P,xi (1) ΔH0 c = − nF( ∂ (E1/2/T) ∂ (1/T))P,xi (2) whe e n =1 and F has i s usual meaning. Fig. 3 shows he E 1/2 s T plo s o Human- and Ho se-Cc, when hey a e immobilized on ei he mixed o pu e hiol monolaye s. In all cases, a linea dec ease o E 1/2 wi h empe a u e was obse ed up o ca. 45 ◦C (Fig. 3), which ansla es in o nega i e ΔS0 c alues (see Table 1). Analogously, he ΔH0 c alues de i ed om E 1/2 /T s. T −1 plo s (Fig. S3) a e also ma kedly nega i e (see Table 1), in ag eemen wi h he expec ed s abiliza ion o he e ous o m due o he s ong Fe(II)-S(Me ) binding and he hyd ophobici y o he heme en i onmen ha a o s i s uncha ged e ous s a e [62–64]. These en opic and en halpic changes a e close o epo ed alues o a a ie y o Cc in solu ion [64,65]. A s aigh o wa d compa ison o he en halpic and en opic con ibu ions o he s anda d po en ial (see Fig. 4) shows he p e alence o he ΔH0 c e m o bo h cy och omes. I is in e es ing o no e ha simila alues o ΔH0 c and ΔS0 c a e ob ained o bo h p o eins, hough hei absolu e alue ends o be smalle o ho se-Cc han o human-Cc as he hiol chain-leng h in he monolaye becomes sho e and he p o eins a e loca ed close o he elec ode su ace. This end keeps he en halpy- –en opy compensa ion upon a ying he hickness o he hiol mono- laye , which p esumably e lec s he sol a ion ea angemen con ibu ion o he ΔH0 c and ΔS0 c alues [66]. 3.2. Elec on ans e kine ics o immobilized Cc S anda d elec on ans e a e cons an s (kS) o he Fe 3+ /Fe 2+ edox con e sion we e de e mined om he a ia ion o he ol am- me ic peak po en ial sepa a ion wi h scan a e, by using a i ing p o- cedu e based on he Bu le Volme o malism wi h a ans e coe icien o 0.5 [67]. Symme ical umpe plo s we e ob ained (see Fig. S4), as expec ed o a well beha ed and quasi- e e sible edox couple. The kS alues de e mined a 25 ◦ C upon immobiliza ion o human- and ho se-Cc on six hiol monolaye s a e plo ed in Fig. 5 as a unc ion o he numbe o me hylene g oups in he molecula backbone o he hiol. Fig. 3. Va ia ion o he midpoin po en ial E 1/2 wi h empe a u e o ho se-Cc (blue and cyan symbols) and human-Cc ( ed and o ange symbols) immobilized on gold elec odes modi ied wi h ei he mixed SAMs ( igh panels) o pu e SAMs (le panels). Solid lines a e linea leas -squa e i s o he da a. The elec oly e solu ion was 20 mM sodium phospha e a pH 7. Fig. 4. En halpic (solid ci cles) and en opic (open ci cles) con ibu ions o he educ ion o ho se-Cc (blue symbols) and human-Cc ( ed symbols) immobilized on o gold elec odes modi ied wi h he indica ed mixed SAMs (uppe panel) o pu e SAMs (lowe panel), as a unc ion o he numbe o me hylene g oups in he hiol hyd oca bon chain. The elec oly e solu ion was 20 mM sodium phospha e a pH 7. J.L. Olloqui-Sa iego e al. Jou nal o Elec oanaly ical Chemis y 981 (2025) 118975 4 Two quali a i e ends a e easily no iceable. Fi s , he a e cons an alues o bo h cy och omes a e highe when hey a e adso bed on mixed COOH:OH-SAMs a he han on pu e COOH-SAMs o he same hickness, as i has been p e iously desc ibed in he case o ho se-Cc [68,69]. Second, he elec on exchange a e o human-Cc is lowe han o ho se- Cc when hey a e adso bed on he same monolaye . I may also be obse ed how bo h p o eins display a cha ac e is ic biphasic a ia ion o k S wi h he monolaye hickness, which has been shown o a ise om a ansi ion om a ic ional con ol o he elec on ans e a e, a small elec ode-p o ein dis ances, o a non-adiaba ic con ol a long elec ode- p o ein dis ances. The e o e, he obse ed beha io o kS as a unc ion o he monolaye hickness can be accoun ed o he ollowing se ial combina ion o ic ional and non-adiaba ic a e cons an s [70,71]: 1 kS=1 kFR +1 kNA =1 kFR +1 k0 NA⋅e−γ(d−d0)=1 kFR +1 k0 NA⋅e−1.12⋅nCH2(3) whe e kFR and kNA a e he ic ional and non-adiaba ic s anda d a e cons an s, espec i ely, d is he elec on ans e dis ance, d0 is a dis- ance o e e ence, k0 NA is he non-adiaba ic s anda d a e cons an a he dis ance d0, γ is he exponen ial dis ance decay unneling pa ame e , which akes a alue o 1.12 pe me hylene uni [72,73], and nCH2 is he numbe o me hylene g oups in he hyd oca bon chain o he hiol monolaye . Sepa a e alues o kFR and k0 NAwe e ob ained by i ing ln kS s nCH2 plo s o Eq. (3). Keeping in mind ha he kFR/k0 NA a io ypically akes small alues (~10 −3 ), he nCH2=0 o dina e in Fig. 5 may be iden i ied wi h he ic ional e m (ln kFR), while he nCH2=10 o dina e is close o i s non- adiaba ic coun e pa (ln kNA). Then, a isual compa ison o he ou plo s in Fig. 5 shows ha (a 25 ◦C) bo h ic ional and non–adiaba ic a e cons an s a e la ge o ho se–Cc han o human–Cc o any gi en monolaye , and ha kine ic di e ences be ween he wo cy och omes dec ease signi ican ly when hey a e adso bed on he less cha ged mixed monolaye s. To p o ide a physical in e p e a ion o he obse ed kine ic ends, kFR and kNA should be exp essed in e ms o he mic oscopic pa ame e s ha cha ac e ize each kine ic egime. Then, ollowing Ma yusho o malism [49], we ob ain: kNA =k0 NAe−γ(d−d0)=Δ0e−γ(d−d0) ℏ 4RT π λ √exp(−λ 4RT)(4) kFR =1 τ s 4RT π λ √exp(−λ 4RT)=1 τ 0 s 4RT π λ √exp(−(λ/4)+Es RT )(5) whe e Δ=Δ0e−γ(d−d0)is he elec onic coupling s eng h be ween he heme g oup and he elec ode, λ is he eo ganiza ion ene gy, τ s is he S okes-shi elaxa ion ime, whose dependence on empe a u e is exp essed as a p oduc o a p e-exponen ial ac o ( τ 0 s) and an expo- nen ial ac i a ion e m (exp(Es/RT)) [34], and all o he symbols ha e hei usual meaning. I should be no ed ha a di ec assessmen o ou esul s in e ms o Eqs. (4) and (5) doesn’ allow us o examine he in- di idual beha io o Δ0, λ, τ 0 s and E s . The e o e, we ha e ex ended ou k s measu emen s o en empe a u es wi hin he 0–45 ◦C ange, so ha plo s o ei he ln (k0 NA/ T √)o ln (kFR/ T √) s.1/T in Fig. 6 lead now o sepa a e es ima es o he ou kine ic pa ame e s o each p o ein and immobiliza ion s eng h, which a e collec ed in Table 2. I may be seen in Table 2 how he alues o he wo kine ic pa am- e e s de e mined in he non-adiaba ic limi , i. e. he eo ganiza ion en- e gy (λ) and he elec on hopping equency a nCH2 =0 (Δ0ℏ−1), a e highe o he mixed monolaye s. Simila esul s ha e been epo ed o ho se-Cc adso bed on pu e HS-(CH 2 ) 15 -COOH and mixed HS-(CH 2 ) 15 - COOH/HS-(CH 2 ) 15 -CH 2 OH monolaye s [30,32]. The Δ0ℏ−1p e-expo- nen ial e m depends on he hickness and elec onic conduc i i y o he in e ening medium be ween he elec ode and he p o ein’s edox cen e , which a e no expec ed o di e signi ican ly o pu e and mixed monolaye s. Analogously, he close simila i y be ween hei ΔH0 c and ΔS0 c alues in Table 1 does no an icipa e di e ences in sol a ion o bond ene gy edox ela ed changes and, he e o e, in he eo ganiza ion ene gy λ alues o he wo ypes o monolaye s. In any case, a de ailed assessmen o hese kine ic esul s would equi e a heo e ical ame- wo k ha accoun s explici ly o he s eng h o he in e ac ion be ween monolaye and p o ein. 3.3. Ra ionaliza ion o elec on ans e kine ics Recen ly, Ma yusho [49] has de eloped a heo e ical model ha accoun s o he in luence o he oscilla o y mo ion o p o eins a ached o hiol monolaye s on he a e o hei elec on exchange wi h he elec ode, encompassing bo h non-adiaba ic and ic ional kine ic limi s. In his model, p o ein oscilla ions ha ake place pe pendicula ly o he elec ode su ace a e assumed o obey an ha monic po en ial cha ac- e ized by a o ce cons an κ. These he mally d i en oscilla ions modi y con inuously he leng h o he elec on ans e pa h, leading o he ollowing exp ession o he non-adiaba ic a e cons an : k0 NA =Δc 0 ℏ 4RT π λc √exp(γ2RT 2κ)exp(−λc 4RT)(6) Fig. 5. Loga i hmic plo s o he s anda d elec on ans e a e cons an o human-Cc (a) and ho se-Cc (b) as a unc ion o he numbe o me hylene g oups pe hiol molecule in he adso bed monolaye s. Ci cles co espond o pu e COOH SAMs, and iangles o mixed COOH:OH SAMs. B oken lines a e leas -squa e i s o Eq. (3), wi h kFR and k0 NA as i ing pa ame e s. Expe imen s we e ca ied ou in 20 mM sodium phospha e bu e o pH 7 and a 25 ◦C. J.L. Olloqui-Sa iego e al. Jou nal o Elec oanaly ical Chemis y 981 (2025) 118975 5 whe e Δc 0 is he elec onic coupling a he equilib ium dis ance o he oscilla ing p o ein, co esponding o nCH2=0, and λc is he co ec ed eo ganiza ion ene gy. I should be no ed ha he Δ0ℏ−1 and λ alues collec ed in Table 1 we e de i ed acco ding o Eq. (4) om he in- e cep s and slopes o Fig. 6b plo s and, acco ding o Eq. (6), hey a e expec ed now o a y wi h he s eng h o he p o ein-monolaye in e ac ion (as measu ed by κ), since a compa ison o Eqs. (4) and (6) shows ha (see he Suppo ing In o ma ion sec ion): Δ0 ℏ≈Δc 0 ℏ λ λc √exp(γ2RTa κ)(7a) and λ≈λc+2(γRTa )2 κ(7b) whe e Ta is he a e age empe a u e in he in e al we ha e analyzed. Eqs. (7a) and (7b) p edic a dec ease o he Δ0/ℏ and λ alues upon inc easing he p o ein immobiliza ion s eng h, un il hey e en ually each hei Δc 0/ℏ and λ c limi ing alues when Ta /κ→0. These heo e ical expec a ions ag ee well wi h he obse ed ends in he kine ic pa am- e e alues collec ed in Table 2 and in p e ious s udies [30,32]. A quan i a i e assessmen o he heo e ical p edic ions can be pe - o med by no ing ha , acco ding o Eqs. (4), (6) and (7), changes in κ co esponding o mixed and pu e hiol monolaye s can be ela ed o hose in k0 NA, Δ0ℏ−1 and λ h ough he ollowing exp essions: (Δ0ℏ−1)mixed (Δ0ℏ−1)pu e = λmixed λpu e √exp(γ2RTa (κ−1 mixed −κ−1 pu e)) = λmixed λpu e √exp(λmixed −λpu e 2RTa )(8a) (k0 NA)mixed (k0 NA)pu e = λmixed λpu e √exp(1 2γ2RTa (κ−1 mixed −κ−1 pu e)) = λmixed λpu e √exp((λmixed −λpu e) 4RTa )(8b) which show clea ly how a weake in e ac ion o he p o ein wi h he mixed monolaye (i. e.κ−1 mixed −κ−1 pu e >0) esul s in highe alues o k0 NA, Δ0ℏ−1 and λ. F om a quan i a i e poin o iew, he obse ed Δ0ℏ−1 a ios o he wo ypes o monolaye s a e 7.2 o human-Cc and 3.3 o ho se Cc, and he obse ed k0 NA a ios a 25 ◦C a e 2.4 o human-Cc and 1.3 o ho se Cc, while he es ima ed alues om he abo e ela ion- ships, wi h λ mixed −λ pu e di e ences ha a e wi hin he con idence in- e als o he λ alues collec ed in Table 2, lie in he ollowing anges: (4.3–9.9) o human-Cc and (2.8–6.5) o ho se Cc in he case o he Δ0ℏ−1 a ios, and (2.2–3.4) o human-Cc and (1.7–2.7) o ho se Cc in he case o he k0 NA a ios, showing a easonable ag eemen be ween he ela ed a ia ions o hopping equency and eo ganiza ion ene gy. A henius-like plo s in Fig. 6a co espond o he ic ional kine ic egime and hei slopes and in e cep s we e analyzed acco ding o Eq. (4), a e inse ing ou p e ious es ima es o λ, o ob ain he ( τ 0 S)−1 and E S alues collec ed in Table 2. I may be seen ha bo h he p e- exponen ial elaxa ion ime and i s associa ed ac i a ion ene gy in- c ease wi h he s eng h o he p o ein/monolaye in e ac ion, as ex- pec ed in he p esence o s onge elec os a ic in e ac ions be ween pep ide esidues and monolaye . Howe e , elaxa ion imes τ S de i ed om he ( τ 0 S)−1 and E S alues in Table 2 lie in he 0.5–10 μ s ange a 298 K and, he e o e, a e much la ge han usual es ima es de i ed ei he om he longi udinal elaxa ion ime o wa e (0.5 ps [75]) o om simula ions o Cy c in aqueous solu ion (800 ps [74]). The abo e disc epancy be ween heo e ically expec ed and expe i- men ally obse ed ic ional ime scales can be econciled in Ma yush- o ’s heo y by conside ing he dynamical esponse o he p o ein in addi ion o ha o he sol en . While he sol en con ibu ion is cha - ac e ized by he S okes-shi elaxa ion ime τ S, he dynamics o he p o ein mo ion a e desc ibed by a cha ac e is ic ime τ γ o ansla ional di usion o e he unneling decay leng h γ −1 . Then, unde condi ions applicable o he elec on exchange be ween a hiol modi ied elec ode and adso bed Cy c [49]: Fig. 6. Loga i hmic plo s o (a) kFRT−1/2 and (b) k0 NAT−1/2 s. T−1 o human-Cc ( ed and o ange symbols) and ho se-Cc (blue and cyan symbols) adso bed on pu e COOH e mina ed SAMs (ci cles) and on mixed COOH:OH e mina ed SAMs. B oken lines a e leas -squa e i s o da a. Expe imen s we e ca ied ou in 20 mM sodium phospha e bu e o pH 7. Table 2 Kine ic pa ame e s desc ibing he a e o elec on exchange be ween a hiol modi ied gold elec ode and human- and ho se-Cc acco ding o eqs. (4) and (5). Monolaye Cc Δ0ℏ−1/ μ s−1( τ 0 S)−1/ μ s−1λ/eV ES/eV Pu e acid Human 150 ±3 7.2 ±0.3 0.29 ± 0.02 0.112 ± 0.007 Pu e acid Ho se 610 ±14 11.2 ±0.5 0.34 ± 0.02 0.094 ± 0.006 Mixed acid Human 1080 ±30 44 ±2 0.40 ± 0.03 0.097 ± 0.006 Mixed acid Ho se 2040 ±50 56 ±3 0.43 ± 0.03 0.086 ± 0.005 J.L. Olloqui-Sa iego e al. Jou nal o Elec oanaly ical Chemis y 981 (2025) 118975 6 kFR =1 τ 0 γ 4RT π λc √exp(−γ2RT κ)exp(−(λc/4)+Eγ RT )(9) whe e τ 0 γ is he p e-exponen ial ac o o τ γ, Eγ i s ac i a ion ene gy and all o he symbols ha e hei p e ious meaning. Now, an e ec i e ic- ional ime τ e can be de ined as: τ e = τ γexp(γ2RT κ)=1 γ2Dcy exp(γ2RT κ)(10) whe e Dcy is he di usion coe icien o Cy c. Assuming ha he di usion coe icien akes i s alue in aqueous solu ion Dcy ≈10−6 cm 2 s −1 [76], and since γ≈1 A −1 [72,73], he obse ed 0.5–10 μ s ange o elaxa ion imes a 298 K, co esponds o a 3.0 ⋅ 10 −3 –2.2 ⋅ 10 −3 eV Å −2 ange o o ce cons an alues. Simila alues (i.e. 2.5 ⋅ 10 −3 –2.4 ⋅ 10 −3 eV Å −2 ) ha e been epo ed o azu in and a Cu A subuni o cy och ome c oxidase physiso bed on pu e and mixed alkane hiol monolaye s [77], while a somewha highe alue o 3.7 ⋅ 10 −3 eV Å −2 can be de i ed o ho se Cy c immobilized on alkane hiol monolaye s by a py idine-Fe coo dina i e liga ion [34]. These o ce cons an alues ha e been shown [49] o be consis en wi h easonable a e age p o ein displace- men s o ~3 Å. I should be no ed again ha he ( τ 0 S)−1 and Es alues collec ed in Table 2 we e de i ed acco ding o Eq. (5) om he in e cep s and slopes o Fig. 6a plo s and, acco ding o Eq. (9), hey a e expec ed o a y wi h he s eng h o he p o ein-monolaye in e ac ion, since (see he Sup- po ing In o ma ion sec ion): Es≈Eγ−3(γRTa )2 2κ(11a) and 1 τ 0 s≈1 τ 0 γ λ λc √exp(−2γ2RTa κ)≈1 τ 0 γ exp(−2γ2RTa κ)(11b) I τ 0 γ and Eγ a e assumed o be independen o he monolaye /p o ein in e ac ion s eng h, eq (11a) co ec ly p edic s an inc ease o Es wi h κ, bu eq (11b) leads o an analogous end o ( τ 0 S)−1, which is con a y o he obse ed esul s in Table 2 and o simple physical expec a ions [78]. While Δc 0/ℏ and λ c can easonably be assumed o be independen o κ in he non-adiaba ic case, he kine ic pa ame e s τ 0 γ and Eγ a e inhe en ly dependen on κ, which limi s he use ulness o Eq. (9) o assess he ole o he immobiliza ion s eng h in he ic ional limi . As i may be obse ed in Fig. 6, and in ag eemen wi h p e ious epo s [44,48], he elec ochemical elec on exchange is as e o ho se-Cc han o human-Cc unde he same immobiliza ion condi ions. Howe e , i is in e es ing o no e ha he a io be ween he alues o any gi en kine ic pa ame e P (whe e P s ands o Δ0ℏ−1, λ, ( τ 0 S)−1 o Es) o he wo p o eins (i. e.: P ho se / P human ) app oaches uni y when he mola ac ion o ca boxylic g oups (x acid ) in he monolaye dec eases, and he SAM-p o ein elec os a ic in e ac ion becomes negligible, as i is illus- a ed in Fig. 7. This quan i a i e end sugges s ha he di e ences in kine ic beha io be ween he wo p o eins a e o igina ed in hei dis inc s uc u al esponse o he elec os a ic pe u ba ion b ough abou by hei in e ac ion wi h he monolaye . A simila conclusion can be eached om he kine ic expe imen s pe o med in homogeneous solu- ion by Rod íguez-Rold´ an e al. [47], whe e he second o de a e con- s an o he elec on ans e be ween a se ies o la ins and ei he ho se-Cc o human-Cc emained he same (wi hin ~ 30 %) o bo h p o eins, hough he i s o de a e cons an o he elec on ans e wi hin he p ecu so complex wi h Cy och ome c oxidase was ou imes highe o ho se-Cc han o human-Cc. Again, ho se-Cc displays as e elec on ans e kine ics only a e in e ac ing wi h a nega i ely cha ged ancho ing si e, while hei elec on ans e kine ics emain essen ially he same in he absence o his ype o elec os a ic pe u ba ion. 4. Conclusions In he p esen wo k we ha e explo ed he he modynamics and ki- ne ics o he in e acial elec on ans e o human-Cc and ho se-Cc adso bed on a a ie y o single and bina y hiol monolaye s. The mo- dynamic pa ame e s do no seem o be a ec ed by he monolaye cha ge densi y, hough edox po en ials and educ ion en opies become somewha mo e posi i e when he elec oac i e p o eins a e immobi- lized on hinne monolaye s and, he e o e, a e loca ed close o he elec ode su ace. Kine ic pa ame e s, on he o he hand, o e a much iche beha io . Thus, elec on ans e a e cons an s o bo h p o eins a e highe when hey a e adso bed on he less cha ged monolaye s, and display a cha ac e is ic biphasic beha io as a unc ion o he monolaye hickness, which co esponds o he ansi ion om he ic ional o he non-adiaba ic kine ic limi s. Highe p e-exponen ial and ac i a ion ac o s a e obse ed in bo h kine ic egimes upon dec easing he numbe o ca boxylic g oups in he monolaye . We ha e shown ha , acco ding o Ma yusho ’s heo y in he non-adiaba ic limi , he appa en hopping equency and eo ganiza ion ene gy de i ed om A henius-like plo s a e expec ed o inc ease as he p o ein/monolaye in e ac ion becomes weake , and ha hei espec i e a ia ions a e quan i a i ely ela ed o each o he , in good ag eemen wi h he expe imen al esul s. Analogous esul s in he ic ional limi we e in e p e ed quali a i ely in e ms o an inc ease o molecula ic ion wi h he s eng h o he elec os a ic in e ac ion be ween p o ein and monolaye . When human-Cc and ho se- Cc elec on ans e kine ics a e compa ed, he ho se a ian always displays as e a es bu , in e es ingly, he alues o he kine ic pa am- e e s o he wo p o eins app oach o each o he as he numbe o ca - boxylic g oups in he monolaye dec eases. This esul sugges s ha he o igin o he di e en elec on ans e a es obse ed lies in di e ences o hei s uc u al lexibili y o accommoda e he con o ma ional changes equi ed o o m he p ecu so complex wi h a nega i ely cha ged edox pa ne . Fig. 7. Loga i hm o he a io be ween kine ic pa ame e alues o ho se-Cc (P ho se ) and human-Cc (P human ) as a unc ion o he mole ac ion o ca boxylic g oups in he monolaye . Pa ame e alues we e aken om Table 2, and hey co espond o: Δ0/ℏ ( ed ci cles), ( τ 0 S)−1 (blue iangles), λ (g een squa es) and Es (black diamonds). J.L. Olloqui-Sa iego e al. Jou nal o Elec oanaly ical Chemis y 981 (2025) 118975 7 CRediT au ho ship con ibu ion s a emen Jos´ e Luis Olloqui-Sa iego: W i ing – o iginal d a , Me hodology, In es iga ion, Funding acquisi ion, Da a cu a ion, Concep ualiza ion. I. M´ a quez: In es iga ion, Da a cu a ion, Concep ualiza ion. Alejand a Gue a-Cas ellano: In es iga ion, Da a cu a ion, Concep ualiza ion. M. Mole o: In es iga ion, Me hodology, Da a cu a ion. Miguel A. De la Rosa: Visualiza ion, Concep ualiza ion, Funding acquisi ion. Juan Jos´ e Cal en e: W i ing – e iew & edi ing, Visualiza ion, Valida ion, Fund- ing acquisi ion, Concep ualiza ion. I ene Díaz-Mo eno: Visualiza ion, Valida ion, Concep ualiza ion, W i ing – e iew & edi ing, Funding acquisi ion. Ra ael And eu: Supe ision, Fo mal analysis, Concep ual- iza ion, Visualiza ion, W i ing – e iew & edi ing. Decla a ion o compe ing in e es The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape . Acknowledgmen s Au ho s hank he inancial suppo by he g an s PID2021–126799NB–I00, PID2021-126663NB-I00, unded by MICIU/ AEI/10.13039/501100011033 and by ERDF/EU, TED2021-130191B- C42 and RED2022-134120-T unded by MCIU/AEI/10.13039/ 501100011033 and ERDF A way o making Eu ope, and Eu opean Union Nex Gene a ionEU/PRTR, Andalusian Go e nmen (BIO-198, US/JUNTA/FEDER; o I.D.-M.) and Ram´ on A eces Founda ion (2021- 2024 o I.D.-M.). This publica ion is pa o he g an POSTD.O. C_21_00395, inanced by he Jun a de Andalucía/CUII and by he ESF+” ( o A.G.-C.). Appendix A. Supplemen a y ma e ial Supplemen a y da a o his a icle can be ound online a h ps://doi. o g/10.1016/j.jelechem.2025.118975. Re e ences [1] G. P´ e ez-Mejías, A. Díaz-Quin ana, A. Gue a-Cas ellano, I. Díaz-Mo eno, M.A. de la Rosa, No el insigh s in o he mechanism o elec on ans e in mi ochond ial cy och ome c, Coo d. Chem. Re . 450 (2022) 214233, h ps://doi.o g/10.1016/j. cc .2021.214233. [2] D. Al a ez-Paggi, L. Hannibal, M.A. Cas o, S. O iedo-Rouco, V. Demicheli, V. T´ o o a, F. Tomasina, R. Radi, D.H. Mu gida, Mul i unc ional cy och ome c: lea ning new icks om an old dog, Chem. Re . 117 (2017) 13382–13460, h ps://doi.o g/10.1021/acs.chem e .7b00257. [3] M. Hü emann, P. Pecina, M. Rainbol , T.H. Sande son, V.E. Kagan, L. Sama a i, J. W. Doan, I. Lee, The mul iple unc ions o cy och ome c and hei egula ion in li e and dea h decisions o he mammalian cell: om espi a ion o apop osis, Mi ochond ion 11 (2011) 369–381, h ps://doi.o g/10.1016/j.mi o.2011.01.010. [4] K. Gonz´ alez-A zola, A. Díaz-Quin ana, N. Be na do-Ga cía, J. Ma ínez-F´ ab egas, F. Ri e o-Rod íguez, M.A. Casado-Comb e as, C.A. Elena-Real, A. Vel´ azquez-C uz, S. Gil-Caballe o, A. Vel´ azquez-Campoy, E. Szulc, M.P. Ga il´ an, I. Ayala, R. A anz, R.M. Ríos, X. Sal a ella, J.M. Valpues a, J.A. He moso, M.A. De la Rosa, I. Díaz- Mo eno, Nucleus- ansloca ed mi ocond ial cy och ome c libe a es nucleophosmin- seques e ed ARF umo supp esso by changing nucleola liquid-liquid phase sepa a ion, Na . S uc . Mol. Biol. 29 (2022) 1024–1036, h ps://doi.o g/10.1038/ s41594-022-00842-3. [5] J. Ma ínez-F´ ab egas, I. Díaz-Mo eno, K. Gonz´ alez-A zola, S. Janochas, J. A. Na a o, M. He ´ as, R. Be nha d , A. Vel´ azquez-Campoy, A. Díaz-Quin ana, M. A. De La Rosa, S uc u al and unc ional analysis o no el human cy och ome c a ge s in apop osis, Mol. Cell. P o eomics 13 (2014) 1439–1456, h ps://doi.o g/ 10.1074/mcp.M113.034322. [6] V.E. Kagan, H.A. Bayi , N.A. Beliko a, O. Kap alo , Y.Y. Tyu ina, V.A. Tyu in, J. Jiang, D.A. S oyano sky, P. Wip , P.M. Kochanek, J.S. G eenbe ge , B. Pi , A. A. Sh edo a, G. Bo isenko, Cy och ome c/ca diolipin ela ions in mi ochond ia: a kiss o dea h, F ee Radic. Biol. Med. 46 (2009) 1439–1453, h ps://doi.o g/ 10.1016/j. ee adbiomed.2009.03.004. [7] J.M. Ga cía-He edia, A. Díaz-Quin ana, M. Salzano, M. O z´ aez, E. P´ e ez-Pay´ a, M. Teixei a, M.A. de La Rosa, I. Díaz-Mo eno, Ty osine phospho yla ion u ns alkaline ansi ion in o a biologically ele an p ocess and makes human cy och ome c beha e as an an i-apop o ic swi ch, J. Biol. Ino g. Chem. 16 (2011) 1155–1168, h ps://doi.o g/10.1007/S00775-011-0804-9. [8] A. Gue a-Cas ellano, A. Díaz-Quin ana, B. Mo eno-Bel ´ an, J. L´ opez-P ados, P. M. Nie o, W. Meis e , J. S a a, M. Teixei a, P. Hildeb and , M.A. De La Rosa, I. Díaz-Mo eno, Mimicking y osine phospho yla ion in human cy och ome c by he e ol ed RNA syn he ase echnique, Chem. A Eu . J. 21 (42) (2015) 15004–15012, h ps://doi.o g/10.1002/chem.201502019. [9] A. Díaz-Quin ana, G. P´ e ez-Mejías, A. Gue a-Cas ellano, M.A. De La Rosa, I. Díaz- Mo eno, Wheel and deal in he mi ochond ial inne memb anes: he ale o cy och ome c and ca diolipin, Oxid. Med. Cell. Longe . (2020) 6813405, h ps:// doi.o g/10.1155/2020/6813405. [10] I. Díaz-Mo eno, A. Vel´ azquez-C uz, S. Cu an-F ench, A. Díaz-Quin ana, M.A. De la Rosa, Nuclea cy och ome c – a mi ochond ial isi o egula ing damaged ch oma in dynamics, FEBS Le . 592 (2018) 172–178, h ps://doi.o g/10.1002/ 1873-3468.12959. [11] I. Díaz-Mo eno, J.M. Ga cía-He edia, A. Díaz-Quin ana, M.A. De La Rosa, Cy och ome c signalosome in mi ochond ia, Eu . Biophys. J. 40 (2011) 1301–1315, h ps://doi.o g/10.1007/s00249-011-0774-4. [12] S. Zaidi, M.I. Hassan, A. Islam, F. Ahmad, The ole o key esidues in s uc u e, unc ion, and s abili y o cy och ome-c, Cell. Mol. Li e Sci. 71 (2014) 229–255, h ps://doi.o g/10.1007/S00018-013-1341-1. [13] S. O iedo-Rouco, J.M. Pe ez-Be oldi, C. Spedalie i, M.A. Cas o, F. Tomasina, V. To o a, R. Radi, D.H. Mu gida, Elec on ans e and con o ma ional ansi ions o cy och ome c a e modula ed by he same dynamical ea u es, A ch. Biochem. Biophys. 680 (2020) 108243, h ps://doi.o g/10.1016/j.abb.2019.108243. [14] M. Paoli, J. Ma les-W igh , A. Smi h, S uc u e- unc ion ela ionships in heme- p o eins, DNA Cell Biol. 21 (2002) 271–280, h ps://doi.o g/10.1089/ 104454902753759690. [15] H. Rode , G.A. El¨ o e, S.W. Englande , S uc u al cha ac e iza ion o olding in e media es in cy och ome c by H-exchange labelling and p o on NMR, Na u e 335 (1988) 700–704, h ps://doi.o g/10.1038/335700A0. [16] L. Hannibal, F. Tomasina, D.A. Capde ila, V. Demicheli, V. T´ o o a, D. Al a ez- Paggi, R. Jemme son, D.H. Mu gida, R. Radi, Al e na i e con o ma ions o cy och ome c: s uc u e, unc ion, and de ec ion, Biochemis y 55 (2016) 407–428, h ps://doi.o g/10.1021/acs.biochem.5b01385. [17] M. Imai, T. Saio, H. Kume a, T. Uchida, F. Inagaki, K. Ishimo i, In es iga ion o he edox-dependen modula ion o s uc u e and dynamics in human cy och ome c, Biochem. Biophys. Res. Commun. 469 (2016) 978–984, h ps://doi.o g/10.1016/j. bb c.2015.12.079. [18] U.A. Zi a e, J. Szus e , M.F. Scocozza, A. Espinoza-Ca a, A.J. Legu o, M. N. Mo gada, A.J. Vila, D.H. Mu gida, The ole o molecula c owding in long- ange me allop o ein elec on ans e : dissec ion in o si e- and sca old-speci ic con ibu ions, Elec ochim. Ac a 294 (2019) 117–125, h ps://doi.o g/10.1016/j. elec ac a.2018.10.069. [19] M. He ´ as, J.A. Na a o, E ec o c owding on he elec on ans e p ocess om plas ocyanin and cy och ome c6 o pho osys em I: a compa a i e s udy om cyanobac e ia o g een algae, Pho osyn h. Res. 107 (2011) 279–286, h ps://doi. o g/10.1007/S11120-011-9637-1. [20] A. Haga man, L. Dui ch, R. Schwei ze -S enne , The con o ma ional mani old o e icy och ome c explo ed by isible and a -UV elec onic ci cula dich oism spec oscopy, Biochemis y 47 (2008) 9667–9677, h ps://doi.o g/10.1021/ BI800729W. [21] J.B. So e , R. Schwei ze -S enne , Nea -exac en halpy-en opy compensa ion go e ns he he mal un olding o p o ona ion s a es o oxidized cy och ome c, J. Biol. Ino g. Chem. 19 (2014) 1181–1194, h ps://doi.o g/10.1007/s00775-014- 1174-x. [22] D.H. Mu gida, P. Hildeb and , He e ogeneous elec on ans e o cy och ome c on coa ed sil e elec odes. Elec ic ield e ec s on s uc u e and edox po en ial, J. Phys. Chem. B 105 (2001) 1578–1586, h ps://doi.o g/10.1021/jp003742n. [23] G. P´ e ez-Mejías, J.L. Olloqui-Sa iego, A. Gue a-Cas ellano, A. Díaz-Quin ana, J. J. Cal en e, R. And eu, M.A. de la Rosa, I. Díaz-Mo eno, Physical con ac be ween cy och ome c1 and cy och ome c inc eases he d i ing o ce o elec on ans e , Biochim. Biophys. Ac a (BBA) - B Bioene ge . 1861 (2020) 148277, h ps://doi. o g/10.1016/j.bbabio.2020.148277. [24] H. Bia a, T. Sch eibe , S. Ka z, J.S. V¨ olle , M. S ola ski, C. Schulz, N. Michael, N. Budisa, J. Kozuch, T. U esch, P. Hildeb and , Long- ange modula ions o elec ic ields in p o eins, J. Phys. Chem. B 122 (2018) 8330–8342, h ps://doi.o g/ 10.1021/acs.jpcb.8b03870. [25] P.M. De Biase, D.A. Paggi, F. Doc o o ich, P. Hildeb and , D.A. Es in, D. H. Mu gida, M.A. Ma i, Molecula basis o he elec ic ield modula ion o cy och ome c s uc u e and unc ion, J. Am. Chem. Soc. 131 (2009) 16248–16256, h ps://doi.o g/10.1021/ja906726n. [26] D. Buh ke, P. Hildeb and , P obing s uc u e and eac ion dynamics o p o eins using ime- esol ed esonance Raman spec oscopy, Chem. Re . 120 (2020) 3577–3630, h ps://doi.o g/10.1021/acs.chem e .9b00429. [27] S. Oelle ich, H. Wacke ba h, P. Hildeb and , Spec oscopic cha ac e iza ion o nonna i e con o ma ional s a es o cy och ome c, J. Phys. Chem. B 106 (2002) 6566–6580, h ps://doi.o g/10.1021/jp013841g. [28] J.M. Ga cía-He edia, I. Díaz-Mo eno, A. Díaz-Quin ana, M. O z´ aez, J.A. Na a o, M. He ´ as, M.A. De la Rosa, Speci ic ni a ion o y osines 46 and 48 makes cy och ome c assemble a non- unc ional apop osome, FEBS Le . 586 (2012) 154–158, h ps://doi.o g/10.1016/j. ebsle .2011.12.007. [29] M.J. Eddowes, H.A.O. Hill, Elec ochemis y o ho se hea cy och ome c, J. Am. Chem. Soc. 101 (1979) (2022) 4461–4464, h ps://doi.o g/10.1021/ja00510a003. [30] S. O iedo-Rouco, C. Spedalie i, M.F. Scocozza, F. Tomasina, V. T´ o o a, R. Radi, D. H. Mu gida, Co ela ed elec ic ield modula ion o elec on ans e pa ame e s J.L. Olloqui-Sa iego e al. Jou nal o Elec oanaly ical Chemis y 981 (2025) 118975 8 and he access o al e na i e con o ma ions o mul i unc ional cy och ome c, Bioelec ochem. 143 (2022), h ps://doi.o g/10.1016/j.bioelechem.2021.107956. [31] D. Al a ez-Paggi, D.F. Ma ín, P.M. Debiase, P. Hildeb and , M.A. Ma í, D. H. Mu gida, Molecula basis o coupled p o ein and elec on ans e dynamics o cy och ome c in biomime ic complexes, J. Am. Chem. Soc. 132 (2010) 5769–5778, h ps://doi.o g/10.1021/ja910707 . [32] D. Al a ez-Paggi, M.A. Cas o, V. T´ o o a, L. Cas o, R. Radi, D.H. Mu gida, Elec os a ically d i en second-sphe e ligand swi ch be ween high and low eo ganiza ion ene gy o ms o na i e cy och ome c, J. Am. Chem. Soc. 135 (2013) 4389–4397, h ps://doi.o g/10.1021/ja311786b. [33] A. K anich, H.K. Ly, P. Hildeb and , D.H. Mu gida, Di ec obse a ion o he ga ing s ep in p o ein elec on ans e : elec ic- ield-con olled p o ein dynamics, J. Am. Chem. Soc. 130 (2008) 9844–9848, h ps://doi.o g/10.1021/ja8016895. [34] H. Yue, D. Khosh a iya, D.H. Waldeck, J. G ochol, P. Hildeb and , D.H. Mu gida, On he elec on ans e mechanism be ween cy och ome c and me al elec odes. E idence o dynamic con ol a sho dis ances, J. Phys. Chem. B 110 (2006) 19906–19913, h ps://doi.o g/10.1021/jp0620670. [35] J.J. Wei, H. Liu, K. Niki, E. Ma goliash, D.H. Waldeck, P obing elec on unneling pa hways: elec ochemical s udy o a hea cy och ome c and i s mu an on py idine- e mina ed SAMs, J. Phys. Chem. B 108 (2004) 16912–16917, h ps:// doi.o g/10.1021/jp048148i. [36] J. Pe o i´ c, R.A. Cla k, H. Yue, D.H. Waldeck, E.F. Bowden, Impac o su ace immobiliza ion and solu ion ionic s eng h on he o mal po en ial o immobilized cy och ome c, Langmui 21 (2005) 6308–6316, h ps://doi.o g/10.1021/ la0500373. [37] D.H. Mu gida, P. Hildeb and , J. Wei, Y.F. He, H. Liu, D.H. Waldeck, Su ace- enhanced esonance aman spec oscopic and elec ochemical s udy o cy och ome c bound on elec odes h ough coo dina ion wi h py idinyl- e mina ed sel - assembled monolaye s, J. Phys. Chem. B 108 (2004) 2261–2269, h ps://doi.o g/ 10.1021/jp0353800. [38] D.A. Paggi, D.F. Ma ín, A. K anich, P. Hildeb and , M.A. Ma í, D.H. Mu gida, Compu e simula ion and SERR de ec ion o cy och ome c dynamics a SAM-coa ed elec odes, Elec ochim. Ac a 54 (2009) 4963–4970, h ps://doi.o g/10.1016/j. elec ac a.2009.02.050. [39] T. Saga a, K. Niwa, A. Sone, K. Niki, C. Hinnen, Redox eac ion mechanism o cy och ome c a modi ied gold elec odes, Langmui 6 (1990) 254–262, h ps:// doi.o g/10.1021/la00091a042. [40] B. Jin, G.X. Wang, D. Millo, P. Hildeb and , X.H. Xia, Elec ic- ield con ol o he pH-dependen edox p ocess o cy och ome c immobilized on a gold elec ode, J. Phys. Chem. C 116 (2012) 13038–13044, h ps://doi.o g/10.1021/jp303740e. [41] L. Wang, D.H. Waldeck, Dena u a ion o cy och ome c and i s pe oxidase ac i i y when immobilized on SAM ilms, J. Phys. Chem. C 112 (2008) 1351–1356, h ps:// doi.o g/10.1021/jp076807w. [42] J.L. Olloqui-Sa iego, G. P´ e ez-Mejías, I. M´ a quez, A. Gue a-Cas ellano, J. J. Cal en e, M.A. de la Rosa, R. And eu, I. Díaz-Mo eno, Elec ic ield-induced unc ional changes in elec ode-immobilized mu an species o human cy och ome c, Biochim. Biophys. Ac a (BBA) – Bioene g. 1863 (2022) 148570, h ps://doi.o g/ 10.1016/j.bbabio.2022.148570. [43] S.C. Fei el, K.R. S iege , A. Kapp, D. Webe , M. Alleg ozzi, M. Piccioli, P. Tu ano, F. Lisda , Insigh s in o in e p o ein elec on ans e o human cy och ome C a ian s a anged in mul ilaye a chi ec u es by means o an a i icial silica nanopa icle ma ix, ACS Omega 1 (2016) 1058–1066, h ps://doi.o g/10.1021/ acsomega.6b00213. [44] H.K. Ly, T. U esch, I. Díaz-Mo eno, J.M. Ga cía-He edia, M.´ A. de La Rosa, P. Hildeb and , Pe u ba ion o he edox si e s uc u e o cy och ome c a ian s upon y osine ni a ion, J. Phys. Chem. B 116 (2012) 5694–5702, h ps://doi.o g/ 10.1021/jp302301m. [45] L. Banci, I. Be ini, J.G. Hube , G.A. Spy oulias, P. Tu ano, Solu ion s uc u e o educed ho se hea cy och ome c, J. Biol. Ino g. Chem. 4 (1999) 21–31, h ps:// doi.o g/10.1007/s007750050285/me ics. [46] S.E. B and, M. Scha lau, L. Ge en, M. Hend ix, C. Pa son, T. Elmendo , E. Neel, K. Pianal o, J. Sil a-Nash, B. Du ham, F. Mille , Accele a ed e olu ion o cy och ome c in highe p ima es, and egula ion o he eac ion be ween cy och ome c and cy och ome oxidase by phospho yla ion, Cells 11 (2022) 4014, h ps://doi.o g/10.3390/cells11244014. [47] V. Rod íguez-Rold´ an, J.M. Ga cía-He edia, J.A. Na a o, M. He ´ as, B. de la Ce da, F.P. Molina-He edia, M.A. de la Rosa, A compa a i e kine ic analysis o he eac i i y o plan , ho se, and human espi a o y cy och ome c owa ds cy och ome c oxidase, Biochem. Biophys. Res. Commun. 346 (2006) 1108–1113, h ps://doi.o g/10.1016/j.bb c.2006.06.022. [48] F. Wege ich, P. Tu ano, M. Alleg ozzi, H. M¨ ohwald, F. Lisda , Elec oac i e mul ilaye assemblies o bili ubin oxidase and human cy och ome C mu an s: insigh in o ma ion and kine ic beha io , Langmui 27 (2011) 4202–4211, h ps://doi.o g/10.1021/la104964z. [49] D.V. Ma yusho , Dynamical e ec s in p o ein elec ochemis y, J. Phys. Chem. B 123 (2019) 7290–7301, h ps://doi.o g/10.1021/acs.jpcb.9b04516. [50] A. Gue a-Cas ellano, I. Díaz-Mo eno, A. Vel´ azquez-Campoy, M.A. de La Rosa, A. Díaz-Quin ana, S uc u al and unc ional cha ac e iza ion o phosphomime ic mu an s o cy och ome c a h eonine 28 and se ine 47, Biochim. Biophys. Ac a – Bioene g. 1857 (2016) 387–395, h ps://doi.o g/10.1016/j.bbabio.2016.01.011. [51] M. Fedu co, Redox eac ions o heme-con aining me allop o eins: dynamic e ec s o sel -assembled monolaye s on he modynamics and kine ics o cy och ome c elec on- ans e eac ions, Coo d. Chem. Re . 209 (2000) 263–331, h ps://doi. o g/10.1016/S0010-8545(00)00292-7. [52] M.T. de G oo , M. Me kx, M.T.M. Kope , Reo ganiza ion o immobilized ho se and yeas cy och ome c induced by pH changes o ni ic oxide binding, Langmui 23 (2007) 3832–3839, h ps://doi.o g/10.1021/la062774k. [53] S. Todo o ic, A.E. Ch is iane, J. Ae, P. Hildeb and , D.H. Mu gida, Con o ma ional ansi ions and edox po en ial shi s o cy och ome P450 induced by immobiliza ion, J. Biol. Ino g. Chem. 11 (2006) 119–127, h ps://doi.o g/ 10.1007/s00775-005-0054-9. [54] M.F. Molinas, A. de Candia, S.H. Szajnman, J.B. Rod íguez, M. Ma í, M. Pe ei a, M. Teixei a, S. Todo o ic, D.H. Mu gida, Elec on ans e dynamics o Rhodo he mus ma inus caa3 cy och ome c domains on biomime ic ilms, PCCP 13 (2011) 18088–18098, h ps://doi.o g/10.1039/c1cp21925a. [55] A. K anich, H. Naumann, F.P. Molina-He edia, H. Jus in Moo e, T. Randall Lee, S. Lecom e, M.A. de la Rosa, P. Hildeb and , D.H. Mu gida, Ga ed elec on ans e o cy och omec6 a biomime ic in e aces: a ime- esol ed SERR s udy, PCCP 11 (2009) 7390–7397, h ps://doi.o g/10.1039/b904434e. [56] C.P. Smi h, H.S. Whi e, Theo y o he in e acial po en ial dis ibu ion and e e sible ol amme ic esponse o elec odes coa ed wi h elec oac i e molecula ilms, Anal. Chem. 64 (20) (1992) 2398–2405, h ps://doi.o g/10.1021/ ac00044a017. [57] P. Ramí ez, A. G ane o, R. And eu, A. Cues a, W.H. Mulde , J.J. Cal en e, Po en ial o ze o cha ge as a sensi i e p obe o he i a ion o ionizable sel -assembled monolaye s, Elec ochem. Commun. 10 (2008) 1548–1550, h ps://doi.o g/ 10.1016/j.elecom.2008.08.004. [58] P. Ramí ez, R. And eu, A. Cues a, C.J. Calzado, J.J. Cal en e, De e mina ion o he po en ial o ze o cha ge o Au(1 1 1) modi ied wi h hiol monolaye s, Anal. Chem. 79 (2007) 6473–6479, h ps://doi.o g/10.1021/ac071341z. [59] J.L. Olloqui-Sa iego, I. M´ a quez, E. F u os-Bel ´ an, I. Díaz-Mo eno, M.A. de La Rosa, J.J. Cal en e, R. And eu, A. Díaz-Quin ana, Key ole o he local hyd ophobici y in he eas pa ch o plas ocyanins on hei he mal s abili y and edox p ope ies, ACS Omega 3 (2018) 11447–11454, h ps://doi.o g/10.1021/ acsomega.8b01612. [60] V.T. Taniguchi, N. Sailasu a-Sco , F.C. Anson, H.B. G ay, The modynamics o me allop o ein elec on ans e eac ions, Pu e Appl. Chem. 52 (1980) 2275–2281, h ps://doi.o g/10.1351/pac198052102275. [61] G. Ba is uzzi, M. Bo sa i, M. Sola, Medium and empe a u e e ec s on he edox chemis y o cy och ome c, Eu . J. Ino g. Chem. (2001) 29893004, h ps://doi.o g/ 10.1002/1099-0682. [62] G. Ba is uzzi, M. Bo sa i, J.A. Cowan, A. Ranie i, M. Sola, Con ol o cy och ome c edox po en ial: axial liga ion and p o ein en i onmen e ec s, J. Am. Chem. Soc. 124 (19) (2002) 5315–5324, h ps://doi.o g/10.1021/ja017479 . [63] M.W. Ma a, R.G. Had , M.E. Reinha d, T. K oll, H. Lim, R.W. Ha sock, R. Alonso- Mo i, M. Cholle , J.M. Glownia, S. Nelson, D. Soka as, K. Kunnus, K.O. Hodgson, B. Hedman, U. Be gmann, K.J. Ga ney, E.I. Solomon, Me allop o ein en a ic con ol o ligand-me al bonds quan i ied by ul a as x- ay spec oscopy, Science 356 (2017) 1276–1280, h ps://doi.o g/10.1126/science.aam6203. [64] G. di Rocco, G. Ba is uzzi, M. Bo sa i, C.A. Bo olo i, A. Ranie i, M. Sola, The en halpic and en opic e ms o he educ ion po en ial o me allop o eins: de e minan s and in e play, Coo d. Chem. Re . 445 (2021) 214071, h ps://doi. o g/10.1016/j.cc .2021.214071. [65] G. Ba is uzzi, M. Bo sa i, G. di Rocco, A. Ranie i, M. Sola, En halpy/en opy compensa ion phenomena in he educ ion he modynamics o elec on anspo me allop o eins, J. Biol. Ino g. Chem. 9 (2004) 23–26, h ps://doi.o g/10.1007/ s00775-003-0490-3. [66] E. G unwald, C. S eel, Sol en eo ganiza ion and he modynamic en halpy—en opy compensa ion, J. Am. Chem. Soc. 117 (1995) 5687–5692, h ps://doi.o g/10.1021/ja00126a009. [67] E. La i on, Gene al exp ession o he linea po en ial sweep ol ammog am in he case o di usionless elec ochemical sys ems, J. Elec oanal. Chem. 101 (1979) 19–28, h ps://doi.o g/10.1016/s0022-0728(79)80075-3. [68] A. El Kasmi, J.M. Wallace, E.F. Bowden, S.M. Bine , R.J. Linde man, Con olling in e acial elec on- ans e kine ics o cy och ome c wi h mixed sel -assembled monolaye s, J. Am. Chem. Soc. 120 (1998) 225–226, h ps://doi.o g/10.1021/ ja973417m. [69] K.L. Da is, B.J. D ews, H. Yue, D.H. Waldeck, K. Kno , R.A. Cla k, Elec on- ans e kine ics o co alen ly a ached cy och ome c/SAM/Au elec ode assemblies, J. Phys. Chem. C 112 (2008) 6571–6576, h ps://doi.o g/10.1021/ jp711834 . [70] D.E. Khosh a iya, J. Wei, H. Liu, H. Yue, D.H. Waldeck, Cha ge- ans e mechanism o cy och ome c adso bed on nanome e hick ilms. Dis inguishing ic ional con ol om con o ma ional ga ing, J. Am. Chem. Soc. 125 (2003) 7704–7714, h ps://doi.o g/10.1021/ja034719 . [71] J.L. Olloqui-Sa iego, B. Mo eno-Bel ´ an, A. Díaz-Quin ana, M.A. de La Rosa, J. J. Cal en e, R. And eu, Tempe a u e-d i en changeo e in he elec on- ans e mechanism o a he mophilic plas ocyanin, J. Phys. Chem. Le . 5 (2014) 910–914, h ps://doi.o g/10.1021/jz500150y. [72] H. Oe e ing, M.N. Paddon- ow, M. Heppene , A.M. Oli e , E. Co sa is, J. W. Ve hoe en, N.S. Hush, Long- ange pho oinduced h ough-bond elec on ans e and adia i e ecombina ion ia igid nonconjuga ed b idges: dis ance and sol en dependence, J. Am. Chem. Soc. 109 (1987) 3258–3269, h ps://doi.o g/ 10.1021/ja00245a014. [73] J.F. Smalley, H.O. Finklea, C.E.D. Chidsey, M.R. Lin o d, S.E. C eage , J.P. Fe a is, K. Chal an , T. Zawodzinsk, S.W. Feldbe g, M.D. New on, He e ogeneous elec on- ans e kine ics o u henium and e ocene edox moie ies h ough alkane hiol monolaye s on gold, J. Am. Chem. Soc. 125 (2003) 2004–2013. J.L. Olloqui-Sa iego e al. Jou nal o Elec oanaly ical Chemis y 981 (2025) 118975 9