Electrochemical studies on c type cytochromes at microelectrodes

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  • 1. Journal of Electroanalytical Chemistry 464 (1999) 76 – 84 Electrochemical studies on c-type cytochromes at microelectrodes M.M. Correia dos Santos a,*, P.M. Paes de Sousa a, M.L. Simoes Goncalves a, H. Lopes b, ˜ ¸ b b I. Moura , J.J.G. Moura a Centro de Quımica Estrutural, Instituto Superior Tecnico, A6. Ro6isco Pais, 1096 Lisboa Codex, Portugal ´ ´ b Departamento de Quımica, Centro de Quımica Fina e Biotecnologia, Faculdade de Ciencias e Tecnologia, Uni6ersidade No6a de Lisboa, ´ ´ ˆ 2825 Monte da Caparica, Portugal Received 23 July 1998; received in revised form 19 October 1998Abstract The aim of this work is to use microelectrodes as a current approach for the study of unmediated electrochemistry of redoxproteins. An electrochemical study of monohemic cytochromes c552 from Pseudomonas nautica 617, cytochrome c553 fromDesulfo6ibrio 6ulgaris and horse heart cytochrome c is presented at inlaid disk microelectrodes of platinum, gold and carbon.Different electrochemical techniques were used such as linear scan, differential pulse and square wave voltammetry. Theelectrochemical response was also analysed at conventional size (macro) electrodes for comparison. In all situations a promoterwas used. The electrochemical behaviour was evaluated in terms of kinetics of the electrode processes and the formal potentialsdetermined. Diffusion coefficients were also calculated from the voltammetric data. A critical comparison of the results obtainedis carried out and the advantages of microelectrodes for electrochemical studies of metalloproteins are pointed out. © 1999Elsevier Science S.A. All rights reserved.Keywords: Cytochrome c552; Cytochrome c553; Horse heart cytochrome c; Electrochemistry; Macro and microelectrodes1. Introduction Several c-type cytochromes are among the metallo- proteins that have been investigated by electrochemical The investigation of electrode reactions of redox methods, since Eddowes and Hill found that essentiallyproteins has attracted widespread interest over past reversible cyclic voltammetry of horse heart cytochromeyears since they constitute a good approach to redox c could be observed at a 4,4%-dipyridyl modified goldprocesses taking place in vivo [1,2]. It is now possible to electrode [3,4]. More recently, direct electrochemistry ofachieve direct electrochemistry of redox proteins with- horse heart cytochrome c has been investigated in theout the need of a mediator between the redox center of presence of various amino acids [5] and at polymer [6]the protein and the electrode. In many circumstances, and cysteine [7] modified gold electrodes. However thethe presence of a promoter may be required, but al- question of how cytochromes function as electronthough such a compound encourages electron transfer transfer proteins is still an up to date subject. Althoughwith the protein to proceed, it does not take part in the the question has been widely addressed by differentelectron transfer process. Under these conditions, a authors, important issues related to molecular recogni-wide range of information can thus be obtained includ- tion, electron transfer mechanisms and conformationaling determination of redox potentials, evaluation of the changes coupled to proton and electron transfer are stillnumber of electrons involved and estimation of the rate a main research topic [8]. Improvements in understand-constants for electron transfer. ing such mechanisms may be expected from compara- * Corresponding author. Fax: + 351-1-846-44-55; e-mail: mcsantos tive studies of a major number of protein systems.@alfa.ist.utl.pt. In most electrochemical studies of metalloproteins0022-0728/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.PII: S 0 0 2 2 - 0 7 2 8 ( 9 8 ) 0 0 4 7 4 - 4
  • 2. M.M. Correia dos Santos et al. / Journal of Electroanalytical Chemistry 464 (1999) 76–84 77conventional size electrodes, metal (e.g. platinum, gold) 6ulgaris cytochrome c553 and P. nautica cytochrome c552as well as non-metal (e.g. carbon), have been used. were purified as described in Refs. [22,25], respectively.However, electrochemistry at microelectrodes (critical 4,4%-Dipyridyl dihydrochloride and 4,4%-dithiodipyridinedimension in the range 0.1 – 50 mm) may present some were purchased from Sigma. All other chemicals usedadvantages over electrodes of conventional size [9]. One were pro-analysis grade and all the solutions were madeof them is the high rate of steady state diffusion due to up with deionized water from a Milli-Q water purifica-enhanced mass transportation by nonplanar diffusion. tion system.So, steady state currents are readily attained and kinetic Protein solutions with concentrations in the rangeparameters may be determined without capacitive cur- 0.04–0.35 mM were prepared in 0.1 M NaNO3 and inrents [10,11]. Moreover, due to their dimensions it is 10 − 2 M phosphate buffer (pH 7.019 0.05). The con-possible to work with minimal size samples, an impor- centrations of the reduced forms of the proteins weretant feature when working with proteins. determined spectrophotometrically using the following The aim of this work is to investigate whether molar absorptivities: m550 = 29500 M − 1 cm − 1 for horsevoltammetric methods using microelectrodes can be an heart cytochrome c [26,27], m553 = 23400 M − 1 cm − 1 foreffective approach for the study of unmediated electro- D. 6ulgaris cytochrome c553 [28] and m552 = 19000 M − 1chemistry of redox proteins. Three c-type cytochromes, cm − 1 for P. nautica cytochrome c552 [22]. All electro-all with the same type of axial ligands at the iron atom, chemical measurements were done in the presence ofi.e. methionyl and histidinyl residues, were chosen: (i) promoters (1 or 15 mM).horse heart cytochrome c, whose electrochemical be- Voltammetric experiments were performed using ahaviour has been widely studied at macroelectrodes potentiostat/galvanostat AUTOLAB/PSTAT10 (with[12 – 16]. This system was recently the subject of a the low current module ecd for measurements withcomparative study at macro and microelectrodes [17] microelectrodes) from Eco-Chemie and the data analy-and its redox behaviour at a large assembly of mi- sis processed by the General Purpose Electrochemicalcroelectrodes was also reported [18 – 20]; (ii) cytochrome System GPES 3.2 software package also from Eco-c553 from Desulfo6ibrio 6ulgaris previously investigated chemie. Different electrochemical techniques were used:by cyclic voltammetry and differential pulse voltamme- linear scan (LS) and cyclic voltammetry (CV), differen-try at macroelectrodes [21] and (iii) cytochrome c552 tial pulse (DP) and square wave (SW) voltammetry. Infrom Pseudomonas nautica 617 isolated from a marine LS scan rates of 2–10 mV s − 1 were used while in CVsediment [22] for which no voltammetric data is the scan rate varied between 10 and 500 mV s − 1. In DPavailable. the pulse amplitude, DE, was 50 mV while the pulse So, in this manuscript, we report the electrochemical duration, tp, was 50 ms. In SW the square wave ampli-behaviour of cytochrome c552 at inlaid disk macro and tude, ESW, was 50 mV, the step height, DESW, was 10microelectrodes of gold, platinum and carbon using mV while the frequency was varied in some experimentsdifferent techniques. Moreover, the electrochemistry of between 8 and 50 Hz. In all experiments, the potentialcytochrome c553 from D. 6ulgaris and horse heart cy- was varied between an initial value Ei and a final valuetochrome c is revisited at microelectrodes of platinum, Ef depending on the redox potential of the cytochromegold and carbon. The redox behaviour of cytochrome under study.c553 is also analysed at a gold macroelectrode. Since the Several working electrodes were tested: Metrohmelectrochemical behaviour of redox proteins at un- macroelectrodes of platinum (Ref. 6.1204.010), goldmodified electrodes is complex and much dependent (Ref. 6.1204.020) and carbon (Ref. 6.1204.000) withupon factors such as the electrode surface conditions diameters of 3.0 and 2.8 mm and a gold electrode withand traces of impurities [2,23,24] in all situations 1.6 mm diameter from Bioanalytical Systems. All mi-throughout this work a promoter is used. The electro- croelectrodes used were purchased from BAS: platinumchemical response is evaluated analysing the reversibil- (Ref. NS-PT25), gold (Ref. NS-AU25) and carbonity of the electrode processes and the formal potentials (Ref. MF-2007) with diameters of 22.0, 20.5 and 6.6determined. Diffusion coefficients are also calculated mm, respectively. Before each experiment (or set offrom the voltammetric data. A critical comparison of experiments) the electrodes were polished using finethe results obtained with macro and microelectrodes is alumina suspension and washed with deionized water,presented and the advantages of microelectrodes for followed by sonication. Immersion in 0.1 M HNO3 waselectrochemical studies of proteins are pointed out. required, sometimes, in order to restore the background signal. The electrode radius was estimated before each set of experiments using the ferro/ferricyanide couple2. Experimental (D=7.84×10 − 6 cm2 s − 1 [29]) in 0.5 M KCl acidified to pH 3. Limiting currents at the rotating disk elec- Horse heart cytochrome c Type VI was obtained trodes were measured for the calculation of thefrom Sigma and used with no further purification. D. macroelectrode radius while steady state currents ob-
  • 3. 78 M.M. Correia dos Santos et al. / Journal of Electroanalytical Chemistry 464 (1999) 76–84Table 1Electrochemical behaviour of monohemic cytochromes at microelectrodesaProtein Electrode Platinum Gold Carbon Technique LS SW DP LS SW DP LS SW DP Parameter/mVP. nautica cytochrome c552 (E3/4−E1/4) or W1/2 65 181 178 68.5 – 177 78 200 197 E1/2 or EP 234 274 281 233 – 283 228 233 249D. 6ulgaris cytochrome c553 (E3/4−E1/4) or W1/2 56 121 101 57 121 102 59 – – E1/2 or EP 9 14 31 11 14 33 4 – –Horse heart cytochrome c (E3/4−E1/4) or W1/2 57 126 96 70 137 118 – – – E1/2 or EP 270 273 290 265 279 282 – – – a Tomes criterion (E3/4−E1/4), peak width at half height (W1/2) and half wave (E1/2) and peak (EP) potentials ( 95 mV).(D =7.84× 10 − 6 cm2 s − 1 [29]) in 0.5 M KCl acidified analysed using different voltammetric methods such asto pH 3. Limiting currents at the rotating disk electrodes linear scan, square wave and differential pulse voltamme-were measured for the calculation of the macroelectrode try. In all experiments the promoter 4,4%-dipyridyl wasradius while steady state currents obtained in linear scan used in a concentration of 15 mM.voltammetry were used to estimate the radius of the Half wave potentials values, E1/2, of LS voltam-microelectrodes. The constancy of the values determined mograms and of peak potentials, EP, of SW and DPsuggests that the polishing between experiments has only voltammograms are summarized in Table 1 for thea small effect on the electrode radius. reduction of the heme proteins at microelectrodes (all In all experiments the auxiliary electrode was a plat- values referred to the SHE). In Fig. 1, LS and SWinum wire and the reference electrode was a saturated voltammograms are shown for the reduction of cy-calomel electrode (potential equal to 245 mV vs. SHE) tochromes c552 and c553 at a platinum microelectrode.or a silver
  • 4. silver chloride electrode (potential equal to Due to the time regime operating at a microelectrode a205 mV vs. SHE). sigmoidal curve characterised by a steady state current, All measurements were done in deaerated solutions ISS, is obtained in LS as long as the potential is scannedwith oxygen free U-type nitrogen and at T =20 9 1°C.3. Results and discussion As is well known, the formal potentials of redoxproteins are very useful in order to understand thebiological reactions in which they may be involved aselectron carriers and voltammetric methods usingmacroelectrodes proved to be valuable tools in supplyingsuch data. However, meaningful thermodynamic poten-tials of metalloproteins require firstly a thorough analysisof the electrochemical results. Marked changes in poten-tial may occur due to, e.g. adsorption phenomena andthe degree of reversibility of the electrode processes. Diagnostic criteria, when using macroelectrodes, arewell established [30]. The situation may be misleading atmicroelectrodes due to the contribution of sphericaldiffusion to mass transport. Steady state and transientvoltammograms are obtained with microelectrodes onlyat both extremes of short and long time scales dependingon the dimension of the electrode [9]. Fig. 1. (A) Linear scan voltammograms (6 = 5 mV s − 1) and (B) square wave voltammograms ( f =8 Hz) for the reduction of (I) 0.22 The reduction of cytochrome c552 from P. nautica 617, mM cytochrome c553 and (II) 0.18 mM cytochrome c552, at a plat-cytochrome c553 from D. 6ulgaris and horse heart cy- inum microelectrode with r =11 mm. Medium: phosphate buffer 0.01tochrome c at the different type of microelectrodes was M (pH 7.0)+0.1 M NaNO3 + 0.015 M 4,4%-dipyridyl.
  • 5. M.M. Correia dos Santos et al. / Journal of Electroanalytical Chemistry 464 (1999) 76–84 79Table 2W1/2 values of differential pulse voltammograms at macro and microelectrodesa W1/2/mV9 5Protein P. nautica D. 6ulgaris b Horse heart cytochrome c552 cytochrome c553 cytochrome cElectrodePtMacro 195 – 91Micro 178 101 96AuMacro 190 94 105Micro 177 102 118CMacro 175 – –Micro 197 – – a Medium: phosphate buffer 0.01 M (pH 7.0) +NaNO3 0.10 M and 4,4%-dipyridyl 0.015 M. b From [17].slowly enough [31]. Depending on the radius of the is the diffusion coefficient, ~ the square wave period andelectrode the sweep rate must be as slow as possible, r the radius of the electrode) which characterises thewithout encountering the irreproducibility that convec- response [32]. Thus they may be well-suited for measure-tive interference causes, but in order to avoid mixed mass ments of formal potentials.transport. Fortunately experimental voltammograms re- Although the net current profiles are bell shaped andmain virtually steady state even when the sweep rate is symmetrical for several values of p, the dimensionlessincreased to values in excess of those one may compute normalised net current, DcP, is a function of p [32]. Thetaking into account the electrode dimensions [31]. This constant value obtained for reversible processes in con-can be easily checked if during the backward scan the ditions of semi-infinite linear diffusion is retained onlycurrent almost retraces that of the forward scan. This for small values of p (large radii or high frequencies). Soretracing feature is characteristic of the steady state and the simplest criterion for the reversible square waveis independent of the kinetics of the electrode process. In response at both macro and microelectrodes is given bySW voltammetry a bell shaped curve is obtained for the the peak width at the half height, W1/2, expressed by [34]:net current no matter what the electrode size [32]. The W1/2 = (RT/nF){3.53+ 3.46x 2 /xSW + 8.1} SW (1)same seems to be true for differential pulse voltammetry. where xSW = nFESW/RT, ESW being the square wave3.1. Re6ersibility analysis amplitude. Experimental values of W1/2, shown in Table 1, for the reduction of cytochrome c553 from D. 6ulgaris Steady state voltammograms were analysed in terms at the Pt and Au microelectrodes and for horse heartof the Tomes criterion [33]. As can be seen from Table cytochrome c at the Pt microelectrode, agree with the1 the reduction of cytochrome c553 at Pt, Au and C theoretical one (123 mV for T= 20°C) within the exper-microelectrodes is reversible. The values found for the imental errors (9 5 mV). The same is not true for theTomes criterion are in quite good agreement with the reduction of cytochrome c552 from P. nautica wheretheoretical ones for a reversible system (56 mV, for larger values are observed, as can be seen in Fig. 1. TheT =20°C [30]). The same behaviour was previously peak due to the reduction of horse heart cytochrome cfound for horse heart cytochrome c at a platinum at the Au electrode is also slightly larger than expectedmicroelectrode though the reduction at a gold microelec- according to a fully reversible process.trode occurs at the boundary of reversibility [17]. In the Differential pulse voltammograms for the reduction ofreduction of c552 from P. nautica the analysis of steady the monohemic cytochromes at macro and microelec-state voltammograms also suggests quasi-reversible be- trodes were also analysed in terms of W1/2. From Tablehaviour. 2 where W1/2 values of differential pulse voltammograms Square wave voltammetry at microelectrodes seems to at both types of electrodes are shown, one may envisagecombine the advantages of both approaches. In particu- that, although no theory has been developed for DPVlar, in sharp contrast with linear scan voltammetry, the with microelectrodes, W1/2 measured with these sensorsvoltammograms are basically invariant in shape over a may give us some insight into the degree of revers-large range of the dimensionless parameter p =4D~/r 2 (D ibility as happens with macroelectrodes. Wider peaks
  • 6. 80 M.M. Correia dos Santos et al. / Journal of Electroanalytical Chemistry 464 (1999) 76–84are obtained at both macro and microelectrodesfor irreversible electrochemical reactions, as previouslychecked by LS and SW voltammetry. On the otherhand W1/2 values close to the width of 91 mV for areversible one-electron reduction process [30] are ob-tained for the reduction of cytochrome c553 at Au andPt microelectrodes and horse heart cytochrome c at thePt electrodes. The above behaviour can also be ob-served in Fig. 2 where differential pulse voltam-mograms at macro and microelectrodes are shown forcytochromes c and c552. Electrochemical studies using a gold macroelectrodewere also performed in D. 6ulgaris cytochrome c553. The Fig. 3. Cyclic voltammograms at a Au macroelectrode with r= 1.4main results obtained at pH 7 are: CV measurements mm for the reduction of 0.034 mM cytochrome c552. Scan rate (mVindicate a reversible behaviour up to scan rates of 500 s − 1): (a) 10; (b) 25; (c) 50; (d) 75; (e) 100. Medium: phosphate buffermV s − 1 with E 0%= E1/2 =4 mV; square wave determi- 0.01 M (pH 7.0)+ 0.1 M NaNO3 +0.015 M 4,4%-dipyridyl.nations lead to E 0%= EP =30 mV and W1/2 =121 mVwhile in DPV E 0% E1/2 =EP +DE/2 =26 mV and 3.2. Determination of the heterogeneous rate constantW1/2 =94 mV were obtained. So this cytochrome has a For those processes that are not reversible the elec-reversible behaviour both at macro and micro- trochemical response contains information regardingelectrodes. the kinetics of the charge transfer reaction. Recently, a As was previously mentioned the ionic medium was simple analysis of quasi-reversible steady state voltam-kept constant throughout the experiments, including mograms was proposed [35] where the heterogeneousthe presence of the promoter. So one must bear in mind charge transfer rate constant, kS, can be found directlythat the experimental values reported are for the from the values of two easily accessible experimentalprotein–promoter–electrode interaction. From the parameters (E1/4 − E1/2) and (E1/2 − E3/4), where E1/2 isvoltammetric behaviour analysed so far, one may say the experimental half-wave potential and E1/4 and E3/4that 4,4%-dipyridyl is as good in promoting electron are voltammetric quartile potentials. The proceduretransfer at inlaid disk microelectrodes as at macroelec- also yields the formal potential E 0%.trodes of identical material. The same may be true for The above methodology was used to extract kineticother substances used as surface modifiers for the pro- information from the steady state voltammograms ob-motion of direct electrochemistry of redox proteins [16]. tained at microdisk electrodes. Cyclic voltammetry at conventional size electrodes was also carried out for comparison since, as is well known, for a quasi- reversible system, kinetic data can be easily determined using Nicholson’s treatment where kS is estimated from the peak potentials separation with increasing scan rates [36]. In Fig. 3 cyclic voltammograms obtained at a gold electrode are shown for cytochrome c552. The reduction of this protein at Pt, Au and C macro- electrodes showed behaviour consistent with quasi- reversible electrochemistry: plots of peak current versus square root of scan rate, 6, were linear up to a certain value after which deviations from linearity became ap- parent, while the peak potential separation increased with 6 (Fig. 3). In Table 3 a summary of the values obtained for the heterogeneous charge transfer rate constant for cy- tochrome c552, together with previous results obtained for horse heart cytochrome c [17], is shown and goodFig. 2. Differential pulse voltammograms (tP = 50 ms) for the reduc- agreement exists between kS values determined at bothtion of (I) 0.20 mM cytochrome c and (II) 0.20 mM cytochrome c552at (A) a platinum macroelectrode with r= 1.5 mm and (B) a platinum macro and microelectrodes. So steady state voltam-microelectrode with r =11 mm. Medium: phosphate buffer 0.01 M mograms recorded using inlaid disk microelectrodes(pH 7.0)+ 0.1 M NaNO3 + 0.015 M 4,4%-dipyridyl. also provide a practical and convenient means to deter-
  • 7. M.M. Correia dos Santos et al. / Journal of Electroanalytical Chemistry 464 (1999) 76–84 81Table 3 agreement with our result.Values of the heterogeneous rate constants, kS, at macro and mi-croelectrodes* 3.3. Calculation of diffusion coefficients 104 kS/cm s−1 Diffusion coefficients, D, of the monohemic cy- aProtein P. nautica Horse heart tochromes were also evaluated from the voltammetric cytochrome c552 cytochrome c data at microelectrodes. Irrespective of the degree ofElectrode reversibility of the electrode reactions steady state cur-Au rents, ISS, are always achieved in linear scan voltamme-Macro 8 91 1309 0.3 try (as long as 6 is small enough as previouslyMicro 491 Not accessible discussed) that are proportional to D [33]:PtMacro 6 91 Reversible ISS = 4nFDcr (2)Micro 4 91 Reversible In square wave voltammetry the situation is not soC straightforward since the net current, DIP, is a functionMacro 50 9 1 – of p =4D~/r 2 as well as of the kinetics of the electrodeMicro 20 9 1 – reaction. For reversible systems, D can be calculated a From [17]. numerically once DIP is known, finding the minimum of * Medium: phosphate buffer 0.01 M (pH 7.0)+NaNO3 0.10 M and the function (DI th − DIp) where the theoretical net cur- p4,4%-dipyridyl 0.015 M. rent DI th is given by: Pmine kinetic parameters of quasi-reversible reactions of D DI th = nFAc Dcp (3) ytp pproteins. As to the accessible range of kS values in transient and the dimensionless net current DcP is a function oftechniques, difficulties arising from charging current p according toand ohmic polarization impose the upper limit. In thesteady state there is no charging current whatsoever, DcP = 0.846 p 1/2 + 1.06+ 0.25 exp (− 0.8 p 1/2) (4)but a upper limit for the accessible kS values is depen-dent upon the dimensions of the electrode. So as to the valid for nDE = 10 mV and nESW = 50 mV [32].reduction of the horse heart cytochrome c at the gold The values thus computed for the diffusion coeffi-microelectrode used, according to the mass transfer cients using this technique, together with those fromparameter m = D/r 10 − 3 cm s − 1 (with D 10 − 6 cm2 steady state voltammograms are shown in Table 4.s − 1), values of kS 10 − 3 cm s − 1 are not accessible for Emphasis should be put on the good agreement amongthe experimental conditions used, unless a smaller elec- the values within the experimental error of 5–10%, eventrode is available. This also agrees with the value in the situations where full reversibility was assumed in(kS 10 − 2 cm s − 1) determined by cyclic voltammetry, the calculations. This was the case for the reduction ofthus showing that the electrode reaction is nearly re- horse heart cytochrome c at a gold microelectrode andversible. Previous electrochemical studies of horse heart cytochrome c552 at a carbon microelectrode wherecytochrome c at a gold macroelectrode in the presence square wave net currents for the lowest frequenciesof 4,4%-dipyridyl showed that the electrode reaction was used produced meaningful values of D.almost reversible with kS =(1.4 to 1.9)× 10 − 2 cm s − 1 So, for horse heart cytochrome c voltammetric dataas determined by ac impedance measurements [37] in at microelectrodes lead to D= (1.79 0.4)× 10 − 6 cm2 s − 1, in good agreement with values determined fromTable 4Diffusion coefficients, D, for monohemic cytochromes estimated from voltammetric techniques with microelectrodesa 106 D/cm2 s−1Electrode Platinum Gold CarbonTechnique LS SW LS SW LS SWProteinP. nautica cytochrome c552 1.0 – 1.0 – 0.8 0.7D. 6ulgaris cytochrome c553 2.0 1.3 1.4 2.1 1.0 –Horse heart cytochrome c 1.2 2.0 1.4 2.0 – – a Medium: phosphate buffer 0.01 M (pH 7.0)+NaNO3 0.10 M and 4,4%-dipyridyl 0.015 M
  • 8. 82 M.M. Correia dos Santos et al. / Journal of Electroanalytical Chemistry 464 (1999) 76–84Table 5Comparison of formal potentials values, E 0% versus SHE, for monohemic cytochromes estimated from voltammetric techniques withmicroelectrodesa E 0%/mV 95Electrode Platinum Gold CarbonTechnique LS SW DP LS SW DP LSProteinP. nautica cytochrome c552 270 – – 265 – – 235D. 6ulgaris cytochrome c553 9 14 6 11 14 8 4Horse heart cytochrome c 270 273 265 265 273 257 - a Medium: phosphate buffer 0.01 M (pH 7.0)+NaNO3 0.10 M and 4,4%-dipyridyl 0.015 M.voltammetric data at macroelectrodes as well as by It is worthwhile to point out that the use of eitherother methods, D = 1.14 × 10 − 6 cm2 s − 1 [38]. For cy- square wave or differential pulse voltammetry at mi-tochromes c553 and c552 diffusion coefficients of (1.69 croelectrodes to estimate formal potentials may be the0.5) × 10 − 6 cm2 s − 1 and (0.99 0.2) × 10 − 6 cm2 s − 1 only chance at very low concentrations of proteins, duewere determined. These values also agree quite well to the low detection limit achieved by both techniques.with those determined from CV when a gold macroelec- Additionally, square wave voltammetry is a faster tech-trode was used: from the slope of the linear region of nique than DPV.the plot peak current versus square root of the scan rate For the reduction of cytochrome c552 from P. nauticaD =1.5× 10 − 6 cm2 s − 1 and D= 1.1 × 10 − 6 cm2 s − 1 that occurs as a quasi-reversible process for the experi-were obtained for cytochrome c553 and c552, respec- mental conditions used throughout this work either E1/2tively. No literature values are known for comparison, or EP potentials are affected by the kinetics of thebut since the molar masses of all the three cytochromes electrode reaction. However, the procedure followed toare alike, similar diffusion coefficients were expected. compute kS from steady state voltammograms also yields the formal potential E 0%= E1/2 + DE 0%, being the correction term DE 0% also found directly from the dif-3.4. Formal potentials E 0% ferences (E1/4 − E1/2) and (E1/2 − E3/4) [35]. From the data shown in Table 5 an average value of E 0%=257 In the light of the above results the formal potentials mV versus SHE was computed. The electrochemicalof the monohemic cytochromes were then estimated responses of P. nautica cytochrome c552 and D. 6ulgarisfrom the voltammetric data at microelectrodes. For the c553 were studied in detail in a wide pH range (5–11) byreversible processes, and assuming the similarity of the cyclic voltammetry at a gold macroelectrode in thediffusion coefficients of both oxidized and reduced presence of 4,4%-dithiodipyridine. As indicated in Fig. 4forms, E1/2 values from steady state voltammograms, as a single pKox for c552 and c553 was determined: 10.60well as EP values from square wave voltammograms, and 10.75, respectively.are a direct measure of E 0% [31,32,39]. As far as DP A wide range of redox potentials were observed forvoltammograms are concerned it looks like that the the heme proteins studied in this work (Table 5). Cy-relation valid for macroelectrodes E1/2 =EP +DE/2 [30] tochrome c553 is a member of the cytochrome c super-still holds for reversible processes at microelectrodes. family. The redox potential of this heme protein isThis can be seen in Table 5 where a summary of the E 0% much more negative than those of other members ofresults obtained in this work is shown. From the good this class. The potential of other cytochromes present inagreement in all situations between E 0% values estimated sulfate reducing bacteria, such as multiheme cy-using either EP or E1/2 data at microelectrodes for horse tochromes (with bis-histidinyl axial ligation) are evenheart cytochrome c and cytochrome c553 from D. 6ul- more negative [25]. The three cytochromes studied heregaris, average values of 26594 mV versus SHE and have the same axial ligands (methionine-histidine). The99 4 mV versus SHE were computed, respectively. 3D X-ray structure of D. 6ulgaris Miyazaki cytochromeThese values are in excellent agreement with previously ˚ c553 was solved at 1.3 A [40]. The structure of ferrocy-determined ones of 25595 mV versus SHE for horse tochrome c553 from D. 6ulgaris Hildenborough washeart cytochrome c [4] and 20 910 mV versus SHE for elucidated by NMR [41]. The heme axial methionylc553 [21] and also with the values reported in this work ligand has the same orientation in D. 6ulgaris Miyazakifor c553 at a gold macroelectrode (20 914 mV versus ferricytochrome c553 [25], in D. 6ulgaris HildenboroughSHE). ferrocytochrome c553 [42], in P. nautica ferrocytochrome
  • 9. M.M. Correia dos Santos et al. / Journal of Electroanalytical Chemistry 464 (1999) 76–84 83c552 [22] and in horse heart ferrocytochrome c [43]. The pH values of the medium is a pK transition associatedoverall structures of both redox states of D. 6ulgaris with an alkaline transition, most probably related tocytochrome c553 are similar, but no 3D structure is yet the exchange of the methionine axial ligand [42] foravailable for P. nautica cytochrome c552 (in progress, in both cytochromes c552 and c553, as can be inferred fromcollaboration with C. Canbillau). Since the redox po- the pKox values.tential of a cytochrome is the result of multiple contri-butions (electrostatic and hydrophobic environment ofthe heme pocket, hydrogen bonding, heme axial ligands 4. Conclusionsgeometry, solvent exposure, etc,…) and assuming thatthe main overall structural features are maintained From the above results microelectrodes proved to bewithin these cases, we may be observing the result of a efficient tools to study the unmediated electrochemicalvery fine tuning of the heme environment in the redox behaviour of redox proteins. Meaningful values for theproperties. Moore and Williams [44] proposed that the formal potentials and diffusion coefficients of theNMR chemical shifts of the methionine methyl reso- proteins under study were obtained in a variety ofnance in ferrocytochromes could be used as an indica- situations: different microelectrodes were used as welltor of the Fe–S bond length (and subtle alterations of as different electrochemical techniques.this parameter). As the bond length shortened, the Steady state currents readily attained due to the verymethyl protons should move further into the cloud of small dimensions of the electrodes make the measure-the heme group experiencing greater shielding and an ment of electrochemical kinetics and transport parame-upper field shift. A correlation was then proposed with ters straightforward procedures. Namely diffusionthe redox potential of monoheme cytochromes with coefficients determined by classical electrochemicalmethionine–histidine coordination. The greater the methods are more susceptible to errors since they lead to a determination of D 1/2 and any error in r, c or I willshift (implying shorter Fe – S bond length) the more be squared when used to calculate the diffusion coeffi-negative is the redox potential. However this proposal cients. On the other hand the use of steady stateseems not to have been further supported by further voltammetry for determining kinetic parameters over-NMR work [45] and the differences in Fe – S bond comes difficulties arising from charging currents andlength may not be the determining factor. The trend ohmic polarization which often plague transient tech-does not seem to apply in this case (chemical shifts of niques. This is particularly true for the quasi-reversiblemethionine methyl group −3.62 ppm c553, − 3.01 ppm reactions for which kS can be easily computed withhorse heart and − 3.50 ppm c552). The redox control is commercially available microelectrodes. Moreover, thea problem not fully understood yet. However it is methodology followed to extract kinetic informationinteresting to note that the main event upon altering the from steady state voltammograms also provides the determination of the formal potentials. Evaluation of these parameters are based on the values of the quartile potentials that are independent of the electrode surface area and the concentration of the protein. The benefits resulting from the use of pulse tech- niques (differential pulse and square wave voltamme- try) are not only the low detection limits achieved. For reversible reactions the formal potentials are also read- ily accessible by measuring peak potentials. Special emphasis is given to the results obtained by differential pulse voltammetry since square wave voltammetry may not be available in older equipment. From the discus- sion of the results it is apparent that although no theory has been developed for DPV with microelectrodes, well known procedures to analyse data obtained with macroelectrodes can be applied to microelectrodes. For non reversible processes, due to the mixed regime oper- ating at microelectrodes, evaluation of kinetic parame- ters and/or correction factors is not straightforward forFig. 4. pH dependence of the redox potentials of D. 6ulgaris cy- pulse techniques. However, depending on the electrodetochrome c553 (upper panel) and P. nautica cytochrome c552 (lowpanel), as determined by cyclic voltammetry measurements at a gold size conditions close to reversibility may be achieved,macroelectrode (r = 1.6 mm). Medium: 0.1 M NaNO3 and 0.001 M which are the best conditions for the determination of4,4%-dithiodipyridine. E 0%.
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