The Fe (III)/Fe(II) redox couple as a probe of immobilized tobacco peroxidase: Effect of the immobilization protocol

Depósi o de in es igación de la Uni e sidad de Se illa h ps://idus.us.es/ “This is an Accep ed Manusc ip o an a icle published by Else ie in Elec ochimica Ac a on Olloqui‐Sa iego, J. L., Zakha o a, G.S., Polozniko , А. А., Cal en e, J. J., Hushpulian, D. M., Go on, L., & And eu, R. (2019). The FE (III)/FE(II) edox couple as a p obe o immobilized obacco pe oxidase: E ec o he immobiliza ion p o ocol. Elec ochimica Ac a, 299, 55-61., a ailable a : h ps://doi.o g/10.1016/j.elec ac a.2018.12.153” 1 The Fe (III) / Fe(II) Redox Couple as a P obe o Immobilized Tobacco Pe oxidase: E ec o he Immobiliza ion P o ocol José Luis Olloqui-Sa iego,a Galina S. Zakha o a,b And ey A. Polozniko ,b Juan José Cal en e,a Dmi y M. Hushpulian,b Lo Go on,c Ra ael And eu a,* a Depa men o Physical Chemis y. Uni e si y o Se illa. P o eso Ga cía González 1. 41012 Se illa. Spain. b D. Rogache cen e o Pedia ic Hema ology, Oncology and Immunology, 1 Samo y Mashela s ., Moscow 117997, Russia. c Depa men o Biochemis y and S uc u al Biology. Lund Uni e si y, P. O. Box 124, 221 00, Lund, Sweden. *Co esponding au ho ’s e-mail: [email p o ec ed] 2 Abs ac Non- u no e ol amme y is a sensi i e ool o cha ac e ize he elec ochemical p ope ies o edox p o eins. Howe e , he ca aly ically compe en oxida ion s a es o mos pe oxidases do no display he equi ed elec ochemical e e sibili y. In his epo , we ci cum en his limi a ion and exploi he ol amme ic esponse associa ed wi h he Fe(III)/Fe(II) edox couple o obacco pe oxidase o p obe he ene ge ics and elec onic connec i i y o he heme pocke . We ha e applied his app oach o a ionalize he p e iously epo ed in luence o he immobiliza ion p o ocol on he elec oca aly ic ac i i y o obacco pe oxidase. To decouple p o on and elec on ans e s eps, measu emen s ha e been ca ied ou o e he 3 ≤ pH ≤ 9 ange and a 1e-/2H+ ladde scheme has been adop ed o hei analysis. A each pH, he modynamic and kine ic pa ame e s associa ed wi h he Fe(III)/Fe(II) edox con e sion we e de e mined as a unc ion o empe a u e in he 0-30ºC ange. Reduc ion en opies and eo ganiza ion ene gies displayed di e en alues o co alen ly immobilized and physiso bed enzymes, poin ing o a la ge in ol emen o he sol en in he las case. These indings, oge he wi h a la ge elec onic coupling be ween he p os he ic g oup and he elec ode, a e indica i e o a pa ial dena u a ion o he physiso bed enzymes as he o igin o hei lowe elec oca aly ic ac i i y. Keywo ds: Tobacco Pe oxidase; P o on Coupled Elec on T ans e ; Fe(III)/Fe(II) Couple; Immobiliza ion P o ocol; The modynamics and Kine ics 3 1. In oduc ion Pe oxidases a e heme enzymes ha ca alyze he oxida ion o a wide a ie y o o ganic and ino ganic subs a es by pe oxides. Thei ca aly ic unc ion in ol es he oxida ion o he heme i on o Compound I, which is hen educed by he subs a e o Compound II and Fe(III) in wo consecu i e elec on ans e s eps [1]. Anionic obacco pe oxidase (TOP) is a class III plan pe oxidase, which has been ecognized as a p omising building block o biosenso s due o i s high s abili y and subs a e speci ici y [2,3]. When pe oxidases a e in eg a ed in media o less elec oca aly ic de ices, such as hi d gene a ion biosenso s, an elec ode eplaces he subs a e as he sou ce o elec ons o educe Compound I and Compound II [4,5]. To gene a e a eliable esponse, his con igu a ion equi es ha elec oac i e enzymes p ese e hei e ia y s uc u e a ound he ca aly ic pocke a e immobiliza ion, and ha edox co ac o s a e adequa ely wi ed o he elec ode su ace [6]. A numbe o s udies ha e desc ibed bo h di ec and media ed elec oca aly ic esponses o obacco pe oxidase adso bed on a a ie y o elec ode su aces [716], bu li le is known on he impac o he enzyme immobiliza ion p o ocol on he edox p ope ies o he heme co ac o . Recen ly, we ha e shown ha he co alen immobiliza ion o c osslinked TOP oligome s (ci-olig-TOP) on a g aphi e elec ode esul s in a signi ican imp o emen o he eco ded elec oca aly ic cu en s, as compa ed o hose p oduced by co alen ly immobilized (ci-mon-TOP) and physically adso bed (pa-mon-TOP) TOP monome s [14]. To explo e he o igin o his beha io , non- u no e ol amme y p o ides a simple and e ec i e me hod o p obe he elec onic wi ing and ene ge ics o he edox cen e o immobilized enzymes [17]. Howe e , his ype o analysis can only be applied o he Fe(III)/Fe(II) edox couple o he heme cen e since he highe oxida ion s a es in ol ed in he ca aly ic cycle o 4 pe oxidases do no p o ide a e e sible ol amme ic esponse unde non- u no e condi ions, excep in he case o cy och ome c pe oxidase [18,19]. Ou p e ious ol amme ic s udy o immobilized TOP was pe o med a pH 7 and 0ºC, whe e he h ee a o emen ioned immobiliza ion p o ocols p oduced simila non- u no e ol ammog ams, in s a k con as wi h hei dis inc elec oca aly ic esponses [14]. In his wo k we ha e ca ied ou a sys ema ic analysis o he in luence o pH and empe a u e on he Fe(III)/Fe(II) edox con e sion o immobilized TOP enzymes. Though only c.a. one i h o he elec oac i e heme g oups o co alen ly immobilized TOP enzymes e ain hei abili y o educe hyd ogen pe oxide ia he Compound I/Compound II/Fe(III) ca aly ic cycle [15], a single and cha ac e is ic Fe(III)/Fe(II) ol amme ic esponse is obse ed o each immobiliza ion p o ocol. To decouple he con ibu ions om p o on and elec on ans e s eps we ha e adop ed a 1e-/2H+ ladde scheme, whe e p o on s eps a e assumed o emain in equilib ium. P o on coupling is shown o lead o a pH dependen con ibu ion o he educ ion en opy o he enzyme, which may help o explain he b oad ange o educ ion en opy alues epo ed in li e a u e o pe oxidases [20]. The e ec o empe a u e on he he modynamic and kine ic cha ge ans e pa ame e s is obse ed o be di e en o physiso bed and chemiso bed enzymes, and delinea es a physical scena io whe e physiso bed enzymes unde go con o ma ional changes ha may explain hei loss o elec oca aly ic ac i i y. 2. Ma e ial and me hods 2.1. Enzyme p oduc ion Wild- ype ecombinan TOP (hyd ogen pe oxide oxido educ ase, EC 1.11.1.7) was exp essed in he o m o inclusion bodies in E. coli BL21(DE3) CodonPlus cells as was 5 desc ibed ea lie [21]. The cons uc ion o he exp ession ec o and he p ocedu e o TOP exp ession, e olding and pu i ica ion a e co e ed in de ail elsewhe e [21]. B ie ly, he biomass om 600 mL o cul u e medium was dis up ed, and he p ecipi a e o inclusion bodies was washed, solubilized in 6 M u ea and added d op by d op o he e olding medium, con aining 1.8 M u ea, 0.1 mM DTT, 0.5 mM oxidized glu a hione, 3 mM СаСl2, 5 µM hemin and 5% glyce ol in 50 mM T is-HCl pH 9.5. A e in i o eac i a ion, -TOP was concen a ed and pu i ied on a Toyopea l HW-55 column. The concen a ion o pe oxidase was measu ed by he abso bance a 403 nm, using he expe imen ally de e mined ex inc ion coe icien alue o 108.0 ± 0.5 mM-1cm-1 o he p o ein monome [22]. The esul ing p epa a ion was homogeneous as judged by SDS- PAGE. The ac i i y o pu i ied TOP owa ds ABTS was abou 4100 U/mg and he RZ alue was 3.0. 2.2. Reagen s 1-E hyl-3-(3-dime hylaminop opyl) ca bodiimide (EDC) and N-hyd oxy sul osuccinimide sodium sal (NHS) we e om Sigma-Ald ich. Sodium phospha e bu e solu ions (SPB) o ixed ionic s eng h we e p epa ed om phospho ic acid, and mono and dihyd ogen sodium phospha e om Fluka, and sodium pe chlo a e and sodium hyd oxide om Sigma-Ald ich. All eagen s we e o high pu i y, and we e used as ecei ed. Wa e was pu i ied wi h a Millipo e Milli-Q sys em ( esis i i y 18 M cm). 2.3. Enzyme immobiliza ion Py oly ic g aphi e elec odes we e cons uc ed by i ing a od o highly o ien ed py oly ic g aphi e om Mine al Technologies in o a PEEK casing, so ha i exposed he edge o he g aphi e planes wi h a ci cula geome ic a ea o 0.07 cm2. P io o enzyme 6 immobiliza ion, g aphi e su aces we e polished wi h ab asi e P2400 sandpape , insed wi h Millipo e wa e and d ied wi h an a gon s eam. To physiso b TOP, a 17 µL d op o 0.11 mg mL-1 TOP in 0.01 M SPB pH 6 was deposi ed on he g aphi e su ace, and physiso p ion om solu ion was allowed o p oceed o 20 h a 4ºC unde we condi ions. Fo co alen coupling o TOP monome s, he elec ode was incuba ed o 2 h a 4ºC in a solu ion o med by mixing a 4.5 µL d op o 20 mM NHS and a 5.5 µL d op o 40 mM EDC, bo h solu ions being also 0.01 M SPB pH 6. A e ac i a ion o he oxidized species a he g aphi e su ace, he elec ode was ho oughly insed wi h wa e and d ied wi h an a gon s eam. Then, in a second incuba ion s ep ha las ed 20 h a 4ºC, he elec ode was capped wi h a 17 µL d op o a 0.11 mg mL-1 TOP solu ion in 0.01 M SPB pH 6. Fo co alen apping o TOP oligome s he elec ode su ace was exposed o a mix u e o h ee d ople s: a 4.5 µL d op o 20 mM NHS, a 5.5 µL d op o 40 mM EDC and 7 µL d op o a 0.26 mg mL-1 TOP solu ion, ha we e also 0.01 M SPB pH 6, o 20 h a 4ºC. I espec i e o he immobiliza ion p o ocol, he enzyme concen a ion in he deposi ion solu ion was 3 M, and he elec odes we e ho oughly insed, i s wi h wa e and hen wi h he wo king bu e solu ion, a e enzyme incuba ion. 2.4. Elec ochemical measu emen s All elec ochemical measu emen s we e pe o med wi h an AUTOLAB PGSTAT 30, om Eco Chemie B.V, in a con en ional h ee elec ode undi ided glass cell, equipped wi h a gas inle and he mos a ed wi h a wa e jacke . The coun e and e e ence 7 elec odes we e a P ba and a AgAgClNaCl sa u a ed elec ode, espec i ely. 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 (23 ± 2ºC) in a non-iso he mal con igu a ion. Repo ed po en ial alues ha e been e e ed o he SHE po en ial scale by adding +192 mV o he expe imen al po en ial alues. All measu emen s we e ca ied ou unde a gon a mosphe e. Wo king solu ions con ained 0.02 M sodium phospha e bu e a he desi ed pH plus sodium pe chlo a e o adjus he ionic s eng h o 0.12 M. 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. 3. Resul s and discussion 3. 1. Impac o Solu ion pH on he Fe(III) / Fe(II) edox con e sion o TOP Figu e 1 illus a es some ol amme ic cha ac e is ics o immobilized TOP unde non- u no e condi ions a 25ºC. A couple o su ace ol amme ic wa es, associa ed wi h he Fe(III)/Fe(II) edox con e sion, can be obse ed a po en ials close o 0 V a pH 3. As he solu ion is made mo e basic, he wa es shi owa ds mo e nega i e po en ials (see Figu e 1a), indica ing ha he edox p ocess is accompanied by p o on exchange wi h solu ion. The su ace concen a ion o elec oac i e heme g oups, as de e mined by in eg a ion o he baseline co ec ed ol ammog ams, was ound o lie wi hin 80  20 pmol cm-2 o ci-olig-TOP and pa-mon-TOP, and wi hin 40  10 pmol cm-2 o ci-mon-TOP. The ull wid h a hal maximum o he peaks a low scan a e ( ypically be ween 150 mV and 200 mV) is la ge han he alue expec ed o a popula ion o iden ical non-in e ac ing edox cen e s (90.6 mV a 25ºC), sugges ing he p esence o a a ie y o en i onmen s a ound he heme g oups, including he possibili y o con o ma ional changes ha may a ec he ca aly ic ac i i y o he enzymes. The 8 peak po en ial sepa a ion p E  emains insensi i e o he po en ial scan a e up o 10 V s-1, which is indica i e o a as elec on exchange be ween p o ein and elec ode [23]. I espec i e o he immobiliza ion p o ocol, he midpoin po en ial 12/ E (de e mined om he a e age o he anodic and ca hodic peak po en ials) a ies linea ly wi h pH in basic solu ions, i eaches a pla eau o pH alues close o 5, and i inc eases again as he solu ion is made mo e acidic (see Figu e 1b). P e iously epo ed 12/ E alues o physiso bed TOP a pH 5 [13] and 7 [14] a e in good ag eemen wi h hose displayed in Figu e 1b. The shape o hese 12/ E s pH cu es is analogous o ha o he po en iome ic i a ion cu es o isoenzymes C and A o ho se adish pe oxidase (HRP), hough hey a e shi ed owa ds mo e acidic pHs and mo e posi i e po en ials in ela ion o hose o HRP-C [24] and HRP-A [25]. The simples eac ion scheme ha can desc ibe he obse ed a ia ion o he midpoin po en ial wi h pH is a six membe ed ladde - scheme (see Scheme 1), in ol ing wo acid/base g oups A1 and A2 whose acid dissocia ion equilib ium cons an s a e dependen on he oxida ion s a e o he heme g oup, and a e deno ed as 1ox, K , 2ox, K , 1 ed , K and 2 ed , K [26,27]. Mo eo e , he h ee p o ona ion s a es o he oxidized heme a e ela ed o hei co esponding educed o ms h ough h ee elec on ans e s eps, each one being cha ac e ized by i s o mal po en ial i E and s anda d a e cons an o elec on ans e s,i k (i = 1, 2, 3). Acco ding o his eac ion scheme, h ee pla eaus co esponding o 1 2 1 / EE , 1 2 2 / EE and 1 2 3 / EE a e o be expec ed in a 12/ E s pH plo when he elec on ans e akes place sequen ially h ough each o he h ee pa allel elec on pa hways, as he solu ion is made mo e basic, acco ding o:         12 1 2 2 12 1 ln 1 ed, ed , HH / ox, ox, HH c / K K / c RT EEFc / K K / c      (1) 15 whe e P and xi s and o p essu e and solu ion composi ion, espec i ely. The slope o he 12/ E s T plo s u ns ou o be he sum o wo con ibu ions: he   2i P,x E / T e m, which is pH independen , and a second pH dependen e m, whose alue e ol es om posi i e o nega i e as he solu ion becomes mo e basic, and i accoun s o he change o slope obse ed in Figu e 3. The   2i P,x E / T e m in eq. (5) is di ec ly ela ed o he educ ion en opy 2 , c S  co esponding o he elec on ans e pa hway 2 in Scheme 1, which can be exp essed in he non-iso he mal cell con igu a ion as [3335]: 2 2 i , c P,x E S nF T       (6) whe e n = 1 and F has i s usual meaning. Analogously, he educ ion en halpy 2 , c H  ( e e ed o he no mal hyd ogen elec ode a oom empe a u e) is gi en by:       2 2 NHE 1 i , c P,x E / T H nF /T        (7) O e he limi ed empe a u e ange conside ed he e, bo h 2 E s T and 2 E / T s 1/T plo s a e linea (see Figu es 4a and b), and he co esponding 2 , c S  and 2 , c H  alues ha e been collec ed in Table 2. These alues lay well wi hin he anges epo ed o pe oxidases in solu ion a pH 7, namely, 210 J mol-1 K-1 ≤ c S  ≤ -120 J mol-1 K-1 and 91 kJ mol-1 K-1 ≤ c H  ≤ -18 kJ mol-1 K-1 [20]. I is in e es ing o no e ha he 2 , c S  and 2 , c H  alues ob ained o ci-olig-TOP and ci-mon-TOP a e simila and nega i e, in s a k con as wi h he posi i e alues de e mined o pa-mon-TOP. In he case o elec on ans e p o eins wi h posi i ely cha ged edox cen e s, mo e posi i e educ ion en opies a e gene ally associa ed wi h an inc ease o he sol en accessibili y o he 16 edox cen e [36]. The e o e, he mo e posi i e 2 , c S  alue o physiso bed TOP enzymes sugges s ha hey acqui e a mo e open and sol en accessible polypep ide s uc u e upon adso p ion on g aphi e. On he o he hand, c osslinking wi h he g aphi e su ace and wi h o he TOP molecules helps o p ese e he na i e and ca aly ically ac i e s uc u e o ci-mon-TOP and ci-olig-TOP, bu i also es ic s he sol en access o he edox cen e . Ex ensi e en halpy-en opy compensa ion, as obse ed in Table 2 o he h ee immobiliza ion p o ocols, poin s also o a subs an ial con ibu ion o he sol en exchange be ween p o ein and solu ion o he ene ge ics o TOP educ ion [37]. 17 Figu e 4. (a) Fo mal po en ial 2 E s absolu e empe a u e T and (b) 2 E / T s 1 T plo s o he h ee indica ed immobiliza ion p o ocols. Solid lines in plo s (a) and (b) a e linea leas - squa es i s. O he expe imen al condi ions: 0.02 M SPB (I = 0.12 M). Table 2. The modynamic and Kine ic Pa ame e s Desc ibing he In luence o Tempe a u e on he Fe(III) / Fe(II) Redox Con e sion o Immobilized TOP. pa-mon-TOP ci-mon-TOP ci-olig-TOP ∆𝑆2,𝑟𝑐 𝑓 / J K-1 mol-1 13 ± 7 –37 ± 7 –24 ± 8 ∆𝐻2,𝑟𝑐 𝑓 / kJ mol-1 2 ± 1 –8 ± 2 –4 ± 1 10−4 ∙ 𝐴2 / s-1 23 ± 8 3.0 ± 0.9 1.7 ± 0.7 ∆𝐻2 ≠ / kJ mol-1 10 ± 1 5.8 ± 0.5 6.2 ± 0.5 Wi hin he empe a u e ange o in e es , he ap s k s pH p o iles we e simila o hose al eady discussed a 25ºC (see Figu e 5), and we e also i ed o eq. (3) o ob ain se s o op imized 1 Q , 2s, k and 3 Q alues a each empe a u e. Bo h 1 Q and 3 Q displayed a s onge a ia ion wi h empe a u e (no shown) han ha o 2s, k , bu we ha e limi ed ou analysis o he 2s, k alues due o he impossibili y o decoupling he con ibu ions o a e and equilib ium cons an s o he obse ed a ia ion o 1 Q and 3 Q wi h empe a u e. 18 Figu e 5. pH and empe a u e dependence o he s anda d elec on ans e a e cons an ap s k o he h ee indica ed immobiliza ion p o ocols. Solid lines a e heo e ical i s compu ed om equa ion (3). O he expe imen al condi ions: 0.02 M SPB (I = 0.12 M). The h ee immobiliza ion p o ocols lead o linea A henius plo s displayed in Figu e 6. The in e cep and slope o hese plo s p o ide he p e-exponen ial ac o ( 2 A ) and ac i a ion en halpy ( 2 ΔH ) o elec on ans e , acco ding o: 2 22 Δ ln ln s, H kA RT   (8) 2 A and 2 ΔH alues a e collec ed in Table 2, and bo h may be seen o dec ease as he ex en o TOP c osslinking inc eases, so ha hei combined alues end o mi iga e he in luence o he immobiliza ion p o ocol on he obse ed a e o elec on ans e . A mo e de ailed in e p e a ion o he 2 A and 2 ΔH alues equi es he adop ion o a speci ic elec on ans e mechanism. The a e o elec on exchange be ween TOP and g aphi e is qui e as , in spi e o he low densi y o elec onic s a es o g aphi e (compa e 0.0022 eV-1 o g aphi e [38,39] wi h 0.28 eV-1 o a ypical me al like gold [40]), which educes he p obabili y o elec on ans e be ween enzyme and elec ode, and ein o ces he possibili y ha his elec on exchange akes place h ough a nonadiaba ic mechanism [40]. The adequacy o 19 his mechanism is also suppo ed by he obse ed insensi i i y o he measu ed ap s k alues o he eplacemen o wa e by a highly iscous solu ion as sol en (speci ically, a 2.8 M glucose solu ion wi h  = 4.9 cP a 22ºC [41]). Then, wi hin he amewo k o a nonadiaba ic elec on ans e egime [41]: 2 2 AB AH (9a) 2 2 λ Δ4 H (9b) whe e AB H and 2  a e he elec onic coupling and eo ganiza ion ene gies, espec i ely. The ex en o elec onic coupling a ies wi h he hickness and elec onic conduc i i y o he in e ening medium be ween elec ode and edox cen e [42]. Since he immobiliza ion p o ocol is no expec ed o induce d ama ic changes in he elec onic conduc i i y o he polypep ide ma ix, he sequence o 2 A alues in Table 2 is likely o e lec di e ences in he dis ance o closes app oach o he heme g oup o he elec ode, which would be hen smalle o pa-mon-TOP han o ci-mon-TOP and ci- olig-TOP. Acco ding o his in e p e a ion, physiso p ion o TOP enzymes would be accompanied by con o ma ional changes ha b ing he heme g oup close o he elec ode su ace. In he case o chemiso bed TOP enzymes, he o ma ion o in e - and in amolecula amide bonds would help o ix he na i e p o ein s uc u e and p e en a close app oach o he heme g oup o he elec ode. 20 Figu e 6. A henius plo o he s anda d elec on ans e a e cons an 2s, k , o he h ee indica ed immobiliza ion p o ocols. Solid lines a e linea leas -squa es i s. O he expe imen al condi ions: 0.02 M SPB (I = 0.12 M). The complex molecula sca old o p o eins es ic s he mobili y o he a ious dipoles (such as pep ide g oups o sol en molecules) ha may eo ien when he cha ge o a edox co ac o is changed in an elec on ans e e en , and he eby i helps o lowe i s eo ganiza ion ene gy in compa ison o simila eac ions in ol ing simple eac an s in solu ion [43]. Mo eo e , he immobiliza ion o a edox p o ein on a su ace is expec ed o educe u he he con ibu ion o he su ounding medium o he eo ganiza ion ene gy [44,45]. Thus, he eo ganiza ion ene gy o cy och ome c has been epo ed o dec ease om 0.6 eV in solu ion down o 0.3 eV in he adso bed s a e [46]. This las alue is close o he eo ganiza ion ene gies de i ed om he 2 H   alues collec ed in Table 2, i. e.: 0.23 eV o ci-mon-TOP, 0.25 eV o ci-olig-TOP and 0.41 eV o physiso bed TOP. The highe alue ob ained o physiso bed TOP is again consis en wi h an easie access o he sol en o he edox cen e and i s in ol emen in 21 he ac i a ion p ocess [47,48], whe eas he lowe alues de e mined o co alen ly immobilized p o eins can be conside ed as a di ec consequence o he mo e igid and igh molecula en elope ha c osslinking gene a es a ound he edox cen e [43]. 3. Conclusions In his wo k we ha e shown how he Fe(III)/Fe(II) edox couple o obacco pe oxidase can be used o p obe he ene ge ics and elec onic connec i i y o he heme pocke . Analysis o he combined e ec s o pH and empe a u e on he ol amme ic esponse leads o a de ailed in e p e a ion o he in luence o he enzyme immobiliza ion p o ocol on i s elec ochemical beha io . As compa ed wi h co alen ly immobilized TOP, physiso bed enzymes a e cha ac e ized by a mo e posi i e educ ion en opy and a highe eo ganiza ion ene gy, which a e indica i e o a mo e ex ensi e sol en in ol emen in he elec on ans e p ocess. These obse a ions, oge he wi h a la ge elec onic coupling be ween heme and elec ode poin o a pa ial dena u a ion o he physo bed TOP enzymes as he o igin o hei low elec oca aly ic ac i i y. The modynamic pa ame e alues a e simila o co alen ly immobilized monome s and oligome s, while a la ge elec onic coupling is esponsible o he somewha highe elec on ans e a e cons an s de e mined o he monome s. Howe e , in e molecula c osslinking esul s in a highe popula ion o elec oac i e oligome s and, o e all, in a mo e ac i e elec oca aly ic ilm. Acknowledgemen s J. L. O., J. J. C. and R. A. acknowledge inancial suppo om he Spanish Minis y o Economy and Compe i i eness and he Eu opean Union FEDER (g an s CTQ2014- 22 52641-P and CTQ2015-71955-REDT (ELECTROBIONET)) and L. G. om he Swedish Resea ch Council (g an 2014-5908). Re e ences [1] H. B. Dun o d, Heme Pe oxidases, John Wiley, Chiches e , 1999. [2] I. G Gaza yan, L. M. Lag imini, Pu i ica ion and Unusual Kine ic P ope ies o a Tobacco Anionic Pe oxidase, Phy ochem. 41 (1996) 1029-1034. [3] I. G. Gaza yan, L. Go on, T. Ruzgas, E. Csö egi, W. Schuhmann, L. M Lag imini, D. M. Khushpulian, V. I. Tishko , Tobacco Pe oxidase as a New Reagen o Ampe ome ic Biosenso s, J. Anal. Chem. 60 (2005) 558–566. [4] C. Lége , P. Be and, Di ec Elec ochemis y o Redox Enzymes as a Tool o Mechanis ic S udies, Chem. Re . 108 (2008) 2379-2438. [5] U. Wollenbe ge , Thi d gene a ion biosenso s – in eg a ing ecogni ion and ansduc ion in elec ochemical senso s. In Biosenso s and Mode n Biospeci ic Analy ical Techniques, L. Go on Ed.; Else ie : Ams e dam, 2005; pp 65-130. [6] L. Be anco , H. R. Lucka i , Enzyme Immobiliza ion o Biological Fuel Cell Applica ions. In Enzyma ic Fuel Cells: om Fundamen als o Applica ions, H. R. Lucka i , P. A anasso and G. R. Johnson Eds.; Wiley: Hoboken, 2014; pp. 208-224. [7] F. D. Mun eanu, A. Lindg en, J. Emnéus, L. Go on, T. Ruzgas, E. Csö egi, A. Ciucu, R. B. Van Huys ee, I. G. Gaza yan, L. M. Lag imini, Bioelec ochemical Moni o ing o Phenols and A oma ic Amines in Flow Injec ion Using No el Plan Pe oxidases, Anal. Chem. 70 (1998) 2596-2600. 23 [8] F. D. Mun eanu, L. Go on, A. Lindg en, T. Ruzgas, J. Emnéus, E. Csö egi, I. G. Gaza yan, I. V. Oupo o , E. A. Ma ee a, L. M. Lag imini, Di ec and Media ed Elec on T ans e Ca alyzed by Anionic Tobacco Pe oxidase, Appl. Biochem. Bio echnol. 88 (2000) 321-333. [9] A. Lindg en, T. Ruzgas, L. Go on, E. Csö egi, G. B. A dila, I. Y. Sakha o , I. G. Gaza yan, Biosenso s Based on No el Pe oxidases wi h Imp o ed P ope ies in Di ec and Media ed Elec on T ans e , Biosens. Bioelec on. 15 (2000) 491–497. [10] S. Gaspa , I. C. Popescu, I. G. Gaza yan, A. G. Bau is a, I. Y. Sakha o , B. Ma iasson, E. Csö egi, Biosenso s Based on No el Plan Pe oxidases: a Compa a i e S udy, Elec ochim. Ac a 46 (2000) 255–264. [11] S. Gaspa , H. Zimme mann, I. G. Gaza yan, E. Csö egi, W. Schuhmann, Hyd ogen Pe oxide Biosenso s Based on Di ec Elec on T ans e om Plan Pe oxidases Immobilized on Sel -Assembled Thiol-Monolaye Modi ied Gold Elec odes, Elec oanalysis 13 (2001) 284-288. [12] E. E. Fe apon o a, J. Cas illo, D. M. Hushpulian, V. I. Tishko , T. Chuba , I. G. Gaza yan, L. Go on, Di ec Elec ochemis y o Recombinan Tobacco Pe oxidase on Gold, Elec ochem. Commun. 7 (2005) 1291–1297. [13] J. Cas illo, E. E. Fe apon o a, D. M. Hushpulian, F. Tasca, V. Tishko , T. Chuba , I. G. Gaza yan, L. Go on, Di ec Elec ochemis y and Bioelec oca alysis o H2O2 Reduc ion o Recombinan Tobacco Pe oxidase on G aphi e. E ec o Pe oxidase Single-poin Mu a ion on Ca2+-modula ed Ca aly ic Ac i i y, J. Elec oanal. Chem. 588 (2006) 112–121. 24 [14] J. L Olloqui-Sa iego, G. S. Zakha o a, A. A. Polozniko , J. J. Cal en e, D. M. Hushpulian, L. Go on, R. And eu, In e p o ein Coupling Enhances he Elec oca aly ic E iciency o Tobacco Pe oxidase Immobilized a a G aphi e Elec ode, Anal. Chem. 87 (2015) 10807-10814. [15] J. L Olloqui-Sa iego, G. S. Zakha o a, A. A. Polozniko , J. J. Cal en e, D. M. Hushpulian, L. Go on, R. And eu, Fen on-like Inac i a ion o Tobacco Pe oxidase Elec oca alysis a Nega i e Po en ials, ACS Ca al. 6 (2016) 7452-7457. [16] P. Bollella, L. Medici, M. Tessema, A. A. Polozniko , D. Hushpulian, V. I. Tishko , R. And eu, D. Leech, N. Mege sa, M. Ma caccio, L. Go on, R. An iochia, Highly Sensi i e, S able and Selec i e Hyd ogen Pe oxide Ampe ome ic Biosenso s Based on Pe oxidases om Di e en Sou ces Wi ed by Os-polyme : A Compa a i e S udy. Solid S a e Ionics 314 (2018) 178-186. [17] F. A. A ms ong, Recen De elopmen s in Dynamic Elec ochemical S udies o Adso bed Enzymes and hei Ac i e Si es, Cu . Opin. Chem. Biol. 9 (2005) 110–117. [18] M. S. Mondal, H. A. Fulle , F. A. A ms ong, Di ec Measu emen o he Reduc ion Po en ial o Ca aly ically Ac i e Cy och ome c Pe oxidase Compound I: Vol amme ic De ec ion o a Re e sible, Coope a i e Two-Elec on T ans e Reac ion, J. Am. Chem. Soc. 118 (1996) 263–264. [19] G. P. S e enson, C.-Y. Lee, G. F. Kennedy, A. Pa kin, R. E. Bake , K. Gillow, F. A. A ms ong, D. J. Ga aghan, A. M. Bond, Theo e ical Analysis o he Two-Elec on T ans e Reac ion ans Expe imen al S udies wi h Su ace-Con ined Cy och ome c Pe oxidase Using La ge-Ampli ude Fou ie T ans o med AC Vol amme y, Langmui 28 (2012) 9864–9877.