- Email: [email protected]
Electrochemistry of c-type cytochromes
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HALAtiJIA,&u+
de Chbtie~ct ~&&chit% Ce&x 3 (France) ._.
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MlRElLLE
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Labotaloj& de .&nie
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&ti&DE&RD
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tJnivcrsi&de
Prmcnce, Place Vicyr. Hugo; 13331
-
:.
..
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BactJr~ettne.C N.&S.. I3274 Marrcille Ced+w 2 (J’rance)
1981: in revised fok
(Rcceivd.19h.Occob&
31st D&mber
1981)
ABST.WCT fiiffsrenttl puke Palnrography~ofIcytachrome&from Desul/ooihrio vulgaris Hildcnborough shows the tixiskncc ‘of a reduction peak at cP_ - -0.20 V (is. Ag/A&I rcfcrcnce el~l-ddc). At the gold ckctrodc and in the ‘prcscncc of 4.4’.blpyri&nc as activating ag&t. n kducti~p_..pc& is observed at E,,= -0.175 V @s.~Ag/&Clj.by differential t&c vol~~metcy -By.cyclic v&mmctr$onc cathodic uhd
one n&di$ peak-@z observed at the gold electrode an4 iri th~~presckc of 4.4’-bipyridinc. The scpnration between tht?se,cwopeak potentials is.apprb~im~~~ly 75 mV. Cyt~h~omc’c& seems to be a relatively fast system hnd a- redo? potentia! value, of. El,, ~0.02--“0.0! v (vs.. normal hydrogen- clcctrodc) obthincd.
When
the.pH
is raised,
the &nching
of.thc
690 n& band
is ~bsirvcd;
has been ii is attrihutcd to chc
disruptioc of t& koti-methionink bond &thk hern6.A @Kvahk oi iO.~~O;l for the quilibrium bctw&n the ‘ttio forms of cytochrome-css3 (ii&~methi&ifie botidcd and iron-methidnine unbondcd heme)’ has been obthined from spectrophotonictric data. .: A: [email protected] cytochron@ ‘cs), +d ~mikxbondrial cyfochr0mc.r suggests that .thc ironmet&io+?e botid.is ~lativ$y.~trpnger in the cast of the bacterial cytochromc q,,.
: INTROtikTION
.’
292
In previous works we have studied ihe electrode. reactions of several- c-type c?tochromes: multiple-heme bacterial cytoehromes from Desulfovibrio [l-3] and Desul/ctronzonus[4] species. and monohcmic mitochondrial horse-heart cytochrome c [5,6]. On the grounds of comparisons between related hemoprtiteins, .the. present study of a bacterial monohemic c-type cytoehrome such as cytoehrome css3 from Desulfovibrio vufguris Hildenborough [7] is very attraetivc. This cytoehrome has a low molar mass (9100). Its primary structure has~been studied by~Bruschi’and Le Gall [S] and an NMR investigation has shown [9] that the heme environment differs from those of other cytoehromes c. Cytoehromc c,,, from another strain of Desuifovibrio vulgaris (Miyazaki) has been described as an electron acceptor for formate dehydrogenase [ 131. Multiple-heme c-type cytochromes studied previously [l-4,1 I, 121 exhibit reversii ble electron-transfer characteristics at the mercury electrode: and.arguments in favor of similar conclusions have also been obtained at the gold electrode. On the other hand. the direct electrochemical reduction of. horse-heart cytoehrome c at the mercury electrode has been shown to take place irreversibly [5,6]; moreover, cytochrome c is inactive at the gold electrode [ 131. It is not out of the question that such behavior may also be observed in the case of cytochrome css3, the proteinic chain of which is sufficiently large to envelop the hemic centre -and thus to delay the electronic exchanges with the electrode. As has been recently reported [14,15], the use of an activating agent such as 4,4’-bipyridine provides a convenient way of obtaining a fast response with horse-heart cytochrome c when using the gold electrode_ This activator is worth testing with cytochrome &. Thus, we describe here voltammetric experiments on the electrode reactions of cytochrome css3 at the mercury and gold electrodes in the absence and in the presence of 4,4’-bipyridine. In the case of horse-heart cytochrome c. it is well known that new conformational states appear when the pH is raised (16,171. In particular, a transition between “neutral’* and “alkaline” state occurs, when the sixth !igand of the hemic iron, a methionine, is replaced by a lysine. This exchange is directly related- to the disappearance of the 695 nm band observed in the absorption spectrum of the oxidized form. The cytoehrome css3 spectrum presents a 690 nm band [7] attributed to an interaction of methionine with heme iron; thus, the disruption of the iron-methionine bond when pH increases has been studied by spectrophotometry in conjunction-with voltammetry measurements. FXPERIMEWTAL
Marerials
Cytoehrome c553 from D. vufgaris Hildenborough was prepared and purified as previously described [S]. The protein was judged to be pure from its spectrum and.by polyacrylamide gel analysis. By using analytical thin-layer gel electrofocusing in polyacrylamide gel (LKB 2117 Multiphor apparatus), .the isoele+iC point of cytochrome css3 was estimated to be 8.0.
.293
.. . .._ :.
For purity co_ntr6ls,we have-lverified-that the absorption spectra of the-sample remained unchanged, hefore]and after voltammetric measurements; a value of 1.1 has.been obttine&for:the ratio; .-
:. i. .. 1..
before and:.a fter:experiment; . ..The:concentration. of the, s$utions lias. ca!culated : after, measuring A&red]: e = 23,400 &f-:i:&n”‘;.;, _; 4,4’.-Bipiridine pu;unt ‘&la;-obutined -from Pluka; all other ~cbemica]s used were reagent grade.‘:_Mercury*as, triply. d&tilJed. Solutions .were -prepared with distilled demineraIiz~d.water. ‘-. ‘1 .._.
bfethods and apparatus
--
..The .working &&&&were either the mercury or the gold electrode. The gold electrode was constructed-in our laboratory by inserting a gold wire into a resin casing. The exposed area was A_-- 0.0079 cm’. The gold surface was polished before each.expe&&nt with uttrafine emery paper until the surface was brought to a mirror finish. The a&liary electrode was a platinum wire and the reference electrode was a Metrohm -silver-silver chloride. (saturated. NaCl solution) electrode (symbolized by Ag/AgCI): Throughout this’ paper, all .potentials are given vs. the Ag/AgCl reference electrode unless otherwise specified. The buffer solution, which also served as supporting electrolyte, was 0.05 M sodium phosphate at pH 7.0. For the study of the effect of pH the (0.05 IW sodium phosphate.+ 0.05 M sodium borate) mixed medium has been used in the pH range 5- 11.5 investigated. _ Oxygen was purged from solutions by bubbling U grade nitrogen for 30 min before the experiment. Temperature was maintained constant at 25 -t 0.05% Differentiai’pulse polarograms. at the dropping mercury electrode were recorded on a Sefram X-Y recorder coupled with a P.A.R. 174A polarographic analyzer equipped with an’ Ml74/70 .drop timer, using a drop time of 5 s, a. pulse amplitude of A4 = - 50 mV and a scan rate of 0.5 mV s- ‘. Differential pulse voltammograms at the gold el%tfoiPe- were recdrdec-.f(oti positive to negative potentials with the same apparatus; using a pulse. rep&ion rate .of 2 s- ’ (i.e. “drop time”= 0.5 s), a pulse amplitude.of A E.==‘- 50 mV and s&n rates of 0.5-2 mV s-‘. Cyclic voltammograms were performed +ith a P.A.R. 175 universal programmer coupled to a P.A.R. I73 potenti‘ostat; using either a Metrohm hanging mercury drop electrode (A =d.022 cm:) -or the-.gold’-;e]e&rode.: A .B.eckman pH-meter. and a Beckman recorder %re. used for pH.measurements at the glass electrode. k Gary 14 spectrophotometer served for all optical measurements. .. . ‘.
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294
RESULTS
Differential pulse polarography and cyclic voltammetty at the mercury electrdde‘
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The differential pulse polarogram of a 150 ,Gkf cytochrome c,,, solution in 0.05 Msodium phosphate buffer (pH 7.0) is shownin Fig. la. One @akat EP = -0.20 V is detected, which is assigned to the reduction of the heme itself. The cyclic.voltammogram obtained at the hanging mercury drop electrode for the same solution by using a scan rate of 5 mV s-r is shown in Fig. 1b. One cathodic peak at Epc- -. -0.2fV and one poorly defined anodic peak at cr,” - - 0.1 V are observed. The anodie peak disappears and the cathodic peak is shrfted toward more negative potentials (E, - -0.38 V at t) = 1 V s-r) by increasing the scan rate. The most favorable.conditions to observe both peaks correspond in fact to the slowest (i-5 mV s-r) scan rates. From the shape and characteristics of differential pulse polarograms‘and cyclic voltammograms, it may be observed that cytochrome csS3 is not a truly reversible electrochemical system at the mercury electrode; moreover, it is to be expected that adsorption phenomena must occur at the mercury surface, as is generally the case with other proteins, for example, cytochrome c 118,191and cytochromes CS [1,2].’
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Fig. 1. Electrochemical behavior at the mercury electrode of 150 phf jtochrome css3 in 0.05 M sodium phosphate buffer pH 7.0: (a) difierential pulse polarograms at.the dropping mercury electwe, wp time 5 s. scan rate 0.5 mV s-I* , (b) cyclic voltanunograms at the hanging mercury drop electrode, scan rate 5 mV s-l. (a - -) Background solutions.
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.;The:~fferentiai-:p;ulsevolt-~~~..of a. IS0 &cytoch.rome zs& solution pH 7.d ~$die gold el&t&le hi thk- &~eiic~+: of ~4,4’~bi&rid& is’.shown in Fig. 2a; a’. .. pborly:.mrirked~~w~~~-at.E__-0+2-V being observed. -gike&se, cathodic and’anodic .. cy@i$ vo@nimetry $x&s inFig. .2b,$&rather shght-. A -: .In .tlie ~&e&de of: O.Oi&I 4,4~bi&idine, substantial enhancements of the- dif-. : ferentid-.ij;ulse.and-,cy&c voltammet~. peaks. arenoted: ,a Gel12shapeddifferential. :pulse vo+mme~ry .pe& at EP A-!& 0.175 V iS detected, -and:well-developed cathodic and I .&,&d. p&s’. -aj+‘-:.+. :+ .0122V and- EPp = -0.14V are observed by cyclic v6ltanu&%y~:Thus,~-ashzis been reported for horse-heart cyttihrome c [14,14], the .activating-effect of 4,4’-bi&idine on the electrode rtiction of cytochrome cSS3is also proved in:. the. present- ease;. The separation of forward’ and reverse peak ’ ltiaJs.AE,.=. E& -7_E+_was found to be.approximately constant (- 75 mV) for the~‘&vest~ scan rates. The cathodid and anoclic peakcurrents, i, and ipo, were found. t& increase -linearly, with n’/*.-(o .T J mV s Y ’ -5 V s 7 I), a. expected for a diffusion-controlled. pro@&.- Moreover,. the. ratio of the peak -currents, i&/i,, is nearly..u&y .in :the range. &restigated. Thus, good arguments in favor of a- rather . reversible. eledtrode-process.in the presentie:of :4;4-bipyridine may. be put forward. As in-&e -czisk of .cyto&rome c; the use -of 4,4’-bipyridine [ 14,151provides a good
41
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(b) .
.
1
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296
T4BLEI Determination
of the heterogeneous rate constant (concentration of cytochrome +,r = 150 PM)
v/v s-1
A Ep /mV
Y,a
k,XIO~/cms-’
0.010 0.020 0.050 0.100 0.200
83 90 105 120 142
1.2 0.8 0.5 0.36 0.25
1.22 I.15 1.14 1.16 1.14
” As defined by Nicholson
,--
._
-
(221.
of obtaining the redox potential of an electrochemically slow hemoprotein by voltammetric measurements. From differential pulse and cyclic voltammetry experiments, we have obtainedE I,z = -0.19 V, i.e. 0.02 f 0.01 V vs. normal hydrogen electrode (this conversion results from measurements of the potential of the Ag/AgCl reference electrode with the hydrogen electrode by using N.B.S. standard buffer solutions [20]), a value which is very close to the redox potential Ei of D. oulgaris cytochrome c553_ It will be noted that this potential is not very different from the value 0.05 * 0.02 V obtained for D. demifzirricans Norway cytochrome cssJ [2 I]. In fact it appears from AEp = Epa - Epc values that cytochrome css3 is not a strictly reversible system. The rate constant for electron transfer can be estimated from the peak potential separations at increasing scan rates [22]. A k, value of about 1 X 10v3 cm s-’ has been obtained by using the Nicholson treatment, which means that the electrode process can be defined as a quasi-reversible one 1231 (see Table 1). way
Effect of pH Differential pulse and cyclic voltammetry peaks with increasing pH are found to be unchanged up to a pH of about 10, then to decrease above this value.. The simultaneous alteration in the absorbance of the 690 nm band in the oxidized form is shown in Fig. 3, indicating- that the structure of cytochrome c,,, is modified; a decrease is also observed for the absorbance of the 529 nm absorption band in the oxidized form. Sharp drops are observed above pH 10 in Fig. 3 on .both absorbance; pH curves. The quenching of the 690 nm band is consistent with the disruption of the iron-methionine bond. It seems that this quenching is also associated with the decrease of the 529 nm band. It may be assumed that a new “alkalke” form of cytochrome css3 app ears: a pK value of 10.9 -C 0.1 for the transition between both, cytochrome cfrs3 forms can be determined from the midpoint pH of both absorbance; -pH curves. This pK value is noticeably higher ‘than. the.pK_ value.(9;1) .&$&ted :ordinarily for the methionine to lysine bonded heme :iransition- of --ho&e-heart:. .
.
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o 9
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6-
. l-7,-:..
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8
1
I
10..
9
. -11
12..PH
Fig. 3. Effect of ;H-on thi absorbance of the 6&I nm-band (1. left-hand scale) and of the 529 nm band (2. right-hand scale) of 150 gM cytocbrome css3 in (0.05 M sodium phosphate+O.OS M sodium borate) buffer.
cytochrome c [ 161.Con&-y ‘to the existence: of an. ‘electroactiie “alkaline” cyto-chrome c form [6], no.other peak has been detected in Qoltammetry experiments-on cytochrome css3by increasing the pH in the potential -range investigated. DISCt.JSSION : ~. -
:
Several -c-type i3ytochromes a& present in- Desulfooibrio species. The typical cytochrome of the genus ~Destilfmibrio..is~the multiple-heme &&chrome c,, but D. uulg&s cyto@rome c5& is a partic&rly iritq&ing h&noprote&: it iS a single-heme pro&i of Imoderate molecular weight,- like -mitochondrial’ cytochrome c, -with the sax& typk<.of _h&mk’:and k&nt attachmejnt of he&e to -.the proteinic chain. CytoChro~tic;,~ has b&n comp+d to’other cytochrom+ c by Dickerson et al. [24], and:@ of $e hemofiioteins’ of this f&nily seerp to be tilosel$ related by evolutionary hqm&gy_:-.
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In&&i, -in. ihi. present.work,: it ,.hti bEti &ablished from differentjal ptilse and dydlic i&m&e&y .$t: the’ go@ :eiectrode in the : presence of 4,4’-bipyridine as activat&ag&t that th& v$ie of.t+&dqx-po@iial EA is very close to 0.02V::A more.‘.k&ativ& -.v$tik’ &.s- b&n dbta@d Iby- Ya& ,.[lO] for D. ~&l&zris~Miyaza&i Ai --pd&&i .&it- ‘69 ~:Stell$a&n cy@chiofle cSs3frtim_,e@ilib~ti~~~ ‘+Fa$u&ke&. [25]; -the-redox potentiai-~~~~heiiie:ih~~d.~~e &+k ~qd:&@rk&&iive. %l!i&:its ~,l . __I-..._ .-
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percentage exposure .to solvent increases. In the case of cytochrome c553, by using the relationship E,’ (V) = -0.015P + 0.345, a percentage exposure P of 22% could’ be predicted. This partial accessibility to the heme could partly explain that, even in the absence of the activating agent 4,4’-bipyridine, a slight current response can. be detected at the gold electrode by differential pulse and cyclic voltammetry. At the mercury electrode, a peak is observed at Et, = -0.20 V (vs. Ag/AgCl, i.e. E, = -0.02 V vs. normal hydrogen electrode), which correponds to a cathodic overpotential of ca. 40 mV. In fact. the exposure of the active centre is not the only factor which governs the kinetics of electronic exchanges at electrodes. A reaction scheme for the electrontransfer reactions of metallo-proteins at electrodes has been proposed by Albery et al. [26]. where a considerable binding energy for the preceding adsorption step seems to be essential. The binding of proteins to the electrode surface has been compared with the bindings to its physiological partners. From kinetic studies on several cytochromes c, Errede and Kamen [27] have shown that the strength of binding is a dominant parameter in reactivity. It appears that this factor must be taken into account to draw a parailel between the behavior on the electrode and the physiological process. The fact that 4,4’-bipyridine acts as an activator agent in the case of both cytochrome c and cytochrome cssj suggests the existence of similar binding interactions and supports the assumption of a prerequisite adsorption step. A comparison between cytochrome cgj3 and horse-heart cytochrome c is of special interest_ Both cytochromes are monohemic, with respective molecular weights .of 9,100 and 13,000. In both cases the axial ligands of the iron atom are the same, i.e. methionine and histidine residues. However, their redox potentials are separated by about 240 mV. The most significant differences have to be sought in sequences and structures. It has been noted by Cookson et al. [9] that for D. ordgaris cytochrome cSS3 there is only limited evidence for structural homology with other c-type cytochromes. The only aromatic amino acids in the cytochrome c,,, sequence are tyrosine residues, and so.me of these tyrosines are far from the heme; it has been suggested that the heme environment is less hydrophobic. The results of our study tend to support the conclusion that the heme environments of horse-heart cytochrome c and D. odgaris cytochrome cSs3 are rather different. Another striking feature is the strong resistance to the effect of increasing BH on the transition between the “neutral” (= iron-methionine bonded heme) and “alkaline” (= iron-methionine unbonded heme) forms. This result suggests that the iron-methionine bond is relatively strong in the case of cytochrome- cssj; and corroborates the fact that a stabilization of Fe(W) state and hence a lower redox potential may be observed with this cytochrome [9]; the ability of methionine to donate electrons is dependent on the protein structure. REFERENCES 1 P. Bianco and J. Haladjian, Biochim. Biophys. Acta, 545 (1979) 86. 2 P. Bianco. G. Fauque and J. Haladjian. Bioelectrochem. Bioenerg.. 6 (1979) 385.
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-4 P.-B&&~&i J; H&&j&n. B&&c&he~n.Bi&snerg.. 8.(1981)-239. .~ :.’ 5. J: Haiadjian, P. B&i& &td P.A.‘Serre, I.- Eiectroaqal. Chem.. 106 (1980) 397., ! --6 P.A- &&;_J::Haladjian @td -P. Bianco, J:Electr~tal. Chem., i22.(i98i) 327. .- .-7. J. Le .Gaii and M. B&&hi-Heriaud;~iri~~K. 0kunuki;M.D. Kamen and J. Sckuzu (Ed%), Structure and : &n&k~f Cy&tm~&$ Ut&$ty of Tokyo Press and University-Park Press, -1968, p. 467, 8 M~B&scM~and_J. Lit Gaili Biochim.=Biophys; Acta, 271 (1972) 48. -9 D-J: Cooksort,~G.R. Moore; RC. Pitt. R.J.P.~_WiBiams. I.D. Campbell. R.P. Ambler. M. Bruschi and J. Le.GaB. Eur. J. B&hem.? 83 (1978) 261:. 10 T. Y&i B&him:_ Biophys.. Aeta., 548 (1979) 96. 1i K. Nii. T:Yagi; H. Inokuchi and KKimura. J. A& Chem. Sot., 101 (1979) 3335. 12 W.F. Sokoi, D.H. Evans, K. N$i and T. Yagi, .T. Elcctroauai. Chem., IO8 (1980) 107. i3 W.R. Heirreman, BJ. Norris and J.F. Goeiz, Anal. Chem., 47 ( 1975) 79. 14 M.f. Eddowes and H.A.G. Hill, J. Am.~Chem. Sot.. 101 (1979) 4461. I5 M.J. Eddowes, H.A.O. HiBand K. Uosaki, Bioeiectrochem. Bioenerg.. 7 (1980) 527. 16 R Dick&on and R. -Timkovich in P.D. Boyer (Ed.), The Enzymes, Vol. 1 I, Academic Press. New York; 1975, p. 397. 17 H. Hasumi, Biochim. Biophys. Acta. 626 (1980) 265. IS F. Scheiler, Bioclectrochem. Bioenerg., 4 (1977) 490. 19 B.A..Kuznetsov, G.P. Shumakovich and N.M. Mestechkina, Bioeiectrochem. Bioenerg.. 4 (1977) 512. 20 R. Bates, Determination of pH. Wiley. New York. 1964. p. 123. 21 G. Fauque, M. Brusbhi and J. Le Gail, Biocherrt. Biophys. Re.s. Commun., 86 (1979) 1020. 22 RS. Nicholson, Anai. Chem, 37 (1965) 1351. 23 A.M. Bond, Modern Polarographic Methods in Analytical Chemistry, Marcel Dekker, New York and Basic;i980, p_ 24. 24 R_E_ Dickerson, R Timkovich and RJ. Almassy. J. Mol. Bioi., 100 (1976) 473. 25 E. Steiiwagen; Nature, 275 (1978) 73. 26 W.J. Aibery, M.J. Eddowes, H.A.0, Hill and A.R. Hiiiman, J. Am. Chem. Sot.. IO3 (1981) 3904. 27 B..Errede and M.D. Kamen, Biochemistry, 17 (1978) 1015.
-_.: __,,j: . :-
. I_._- .: .:-.r--~-. .. ~. _,.. ;. . : ._._,:r-. _ ~.
-Jtiti .I-’
PIE'I+BIAN& Lbo&& M&eille
.‘.
HALAtiJIA,&u+
de Chbtie~ct ~&&chit% Ce&x 3 (France) ._.
:
:
MlRElLLE
....~
.-
BRUSCHI
Labotaloj& de .&nie
.:
.. -.
-.
_,__,
.. :. :
...~
.,
:
&ti&DE&RD
des Ctm~lcx&
._. _. . .. .
tJnivcrsi&de
Prmcnce, Place Vicyr. Hugo; 13331
-
:.
..
-. ._
BactJr~ettne.C N.&S.. I3274 Marrcille Ced+w 2 (J’rance)
1981: in revised fok
(Rcceivd.19h.Occob&
31st D&mber
1981)
ABST.WCT fiiffsrenttl puke Palnrography~ofIcytachrome&from Desul/ooihrio vulgaris Hildcnborough shows the tixiskncc ‘of a reduction peak at cP_ - -0.20 V (is. Ag/A&I rcfcrcnce el~l-ddc). At the gold ckctrodc and in the ‘prcscncc of 4.4’.blpyri&nc as activating ag&t. n kducti~p_..pc& is observed at E,,= -0.175 V @s.~Ag/&Clj.by differential t&c vol~~metcy -By.cyclic v&mmctr$onc cathodic uhd
one n&di$ peak-@z observed at the gold electrode an4 iri th~~presckc of 4.4’-bipyridinc. The scpnration between tht?se,cwopeak potentials is.apprb~im~~~ly 75 mV. Cyt~h~omc’c& seems to be a relatively fast system hnd a- redo? potentia! value, of. El,, ~0.02--“0.0! v (vs.. normal hydrogen- clcctrodc) obthincd.
When
the.pH
is raised,
the &nching
of.thc
690 n& band
is ~bsirvcd;
has been ii is attrihutcd to chc
disruptioc of t& koti-methionink bond &thk hern6.A @Kvahk oi iO.~~O;l for the quilibrium bctw&n the ‘ttio forms of cytochrome-css3 (ii&~methi&ifie botidcd and iron-methidnine unbondcd heme)’ has been obthined from spectrophotonictric data. .: A: [email protected] cytochron@ ‘cs), +d ~mikxbondrial cyfochr0mc.r suggests that .thc ironmet&io+?e botid.is ~lativ$y.~trpnger in the cast of the bacterial cytochromc q,,.
: INTROtikTION
.’
292
In previous works we have studied ihe electrode. reactions of several- c-type c?tochromes: multiple-heme bacterial cytoehromes from Desulfovibrio [l-3] and Desul/ctronzonus[4] species. and monohcmic mitochondrial horse-heart cytochrome c [5,6]. On the grounds of comparisons between related hemoprtiteins, .the. present study of a bacterial monohemic c-type cytoehrome such as cytoehrome css3 from Desulfovibrio vufguris Hildenborough [7] is very attraetivc. This cytoehrome has a low molar mass (9100). Its primary structure has~been studied by~Bruschi’and Le Gall [S] and an NMR investigation has shown [9] that the heme environment differs from those of other cytoehromes c. Cytoehromc c,,, from another strain of Desuifovibrio vulgaris (Miyazaki) has been described as an electron acceptor for formate dehydrogenase [ 131. Multiple-heme c-type cytochromes studied previously [l-4,1 I, 121 exhibit reversii ble electron-transfer characteristics at the mercury electrode: and.arguments in favor of similar conclusions have also been obtained at the gold electrode. On the other hand. the direct electrochemical reduction of. horse-heart cytoehrome c at the mercury electrode has been shown to take place irreversibly [5,6]; moreover, cytochrome c is inactive at the gold electrode [ 131. It is not out of the question that such behavior may also be observed in the case of cytochrome css3, the proteinic chain of which is sufficiently large to envelop the hemic centre -and thus to delay the electronic exchanges with the electrode. As has been recently reported [14,15], the use of an activating agent such as 4,4’-bipyridine provides a convenient way of obtaining a fast response with horse-heart cytochrome c when using the gold electrode_ This activator is worth testing with cytochrome &. Thus, we describe here voltammetric experiments on the electrode reactions of cytochrome css3 at the mercury and gold electrodes in the absence and in the presence of 4,4’-bipyridine. In the case of horse-heart cytochrome c. it is well known that new conformational states appear when the pH is raised (16,171. In particular, a transition between “neutral’* and “alkaline” state occurs, when the sixth !igand of the hemic iron, a methionine, is replaced by a lysine. This exchange is directly related- to the disappearance of the 695 nm band observed in the absorption spectrum of the oxidized form. The cytoehrome css3 spectrum presents a 690 nm band [7] attributed to an interaction of methionine with heme iron; thus, the disruption of the iron-methionine bond when pH increases has been studied by spectrophotometry in conjunction-with voltammetry measurements. FXPERIMEWTAL
Marerials
Cytoehrome c553 from D. vufgaris Hildenborough was prepared and purified as previously described [S]. The protein was judged to be pure from its spectrum and.by polyacrylamide gel analysis. By using analytical thin-layer gel electrofocusing in polyacrylamide gel (LKB 2117 Multiphor apparatus), .the isoele+iC point of cytochrome css3 was estimated to be 8.0.
.293
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For purity co_ntr6ls,we have-lverified-that the absorption spectra of the-sample remained unchanged, hefore]and after voltammetric measurements; a value of 1.1 has.been obttine&for:the ratio; .-
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before and:.a fter:experiment; . ..The:concentration. of the, s$utions lias. ca!culated : after, measuring A&red]: e = 23,400 &f-:i:&n”‘;.;, _; 4,4’.-Bipiridine pu;unt ‘&la;-obutined -from Pluka; all other ~cbemica]s used were reagent grade.‘:_Mercury*as, triply. d&tilJed. Solutions .were -prepared with distilled demineraIiz~d.water. ‘-. ‘1 .._.
bfethods and apparatus
--
..The .working &&&&were either the mercury or the gold electrode. The gold electrode was constructed-in our laboratory by inserting a gold wire into a resin casing. The exposed area was A_-- 0.0079 cm’. The gold surface was polished before each.expe&&nt with uttrafine emery paper until the surface was brought to a mirror finish. The a&liary electrode was a platinum wire and the reference electrode was a Metrohm -silver-silver chloride. (saturated. NaCl solution) electrode (symbolized by Ag/AgCI): Throughout this’ paper, all .potentials are given vs. the Ag/AgCl reference electrode unless otherwise specified. The buffer solution, which also served as supporting electrolyte, was 0.05 M sodium phosphate at pH 7.0. For the study of the effect of pH the (0.05 IW sodium phosphate.+ 0.05 M sodium borate) mixed medium has been used in the pH range 5- 11.5 investigated. _ Oxygen was purged from solutions by bubbling U grade nitrogen for 30 min before the experiment. Temperature was maintained constant at 25 -t 0.05% Differentiai’pulse polarograms. at the dropping mercury electrode were recorded on a Sefram X-Y recorder coupled with a P.A.R. 174A polarographic analyzer equipped with an’ Ml74/70 .drop timer, using a drop time of 5 s, a. pulse amplitude of A4 = - 50 mV and a scan rate of 0.5 mV s- ‘. Differential pulse voltammograms at the gold el%tfoiPe- were recdrdec-.f(oti positive to negative potentials with the same apparatus; using a pulse. rep&ion rate .of 2 s- ’ (i.e. “drop time”= 0.5 s), a pulse amplitude.of A E.==‘- 50 mV and s&n rates of 0.5-2 mV s-‘. Cyclic voltammograms were performed +ith a P.A.R. 175 universal programmer coupled to a P.A.R. I73 potenti‘ostat; using either a Metrohm hanging mercury drop electrode (A =d.022 cm:) -or the-.gold’-;e]e&rode.: A .B.eckman pH-meter. and a Beckman recorder %re. used for pH.measurements at the glass electrode. k Gary 14 spectrophotometer served for all optical measurements. .. . ‘.
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RESULTS
Differential pulse polarography and cyclic voltammetty at the mercury electrdde‘
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The differential pulse polarogram of a 150 ,Gkf cytochrome c,,, solution in 0.05 Msodium phosphate buffer (pH 7.0) is shownin Fig. la. One @akat EP = -0.20 V is detected, which is assigned to the reduction of the heme itself. The cyclic.voltammogram obtained at the hanging mercury drop electrode for the same solution by using a scan rate of 5 mV s-r is shown in Fig. 1b. One cathodic peak at Epc- -. -0.2fV and one poorly defined anodic peak at cr,” - - 0.1 V are observed. The anodie peak disappears and the cathodic peak is shrfted toward more negative potentials (E, - -0.38 V at t) = 1 V s-r) by increasing the scan rate. The most favorable.conditions to observe both peaks correspond in fact to the slowest (i-5 mV s-r) scan rates. From the shape and characteristics of differential pulse polarograms‘and cyclic voltammograms, it may be observed that cytochrome csS3 is not a truly reversible electrochemical system at the mercury electrode; moreover, it is to be expected that adsorption phenomena must occur at the mercury surface, as is generally the case with other proteins, for example, cytochrome c 118,191and cytochromes CS [1,2].’
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Fig. 1. Electrochemical behavior at the mercury electrode of 150 phf jtochrome css3 in 0.05 M sodium phosphate buffer pH 7.0: (a) difierential pulse polarograms at.the dropping mercury electwe, wp time 5 s. scan rate 0.5 mV s-I* , (b) cyclic voltanunograms at the hanging mercury drop electrode, scan rate 5 mV s-l. (a - -) Background solutions.
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T4BLEI Determination
of the heterogeneous rate constant (concentration of cytochrome +,r = 150 PM)
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0.010 0.020 0.050 0.100 0.200
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” As defined by Nicholson
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of obtaining the redox potential of an electrochemically slow hemoprotein by voltammetric measurements. From differential pulse and cyclic voltammetry experiments, we have obtainedE I,z = -0.19 V, i.e. 0.02 f 0.01 V vs. normal hydrogen electrode (this conversion results from measurements of the potential of the Ag/AgCl reference electrode with the hydrogen electrode by using N.B.S. standard buffer solutions [20]), a value which is very close to the redox potential Ei of D. oulgaris cytochrome c553_ It will be noted that this potential is not very different from the value 0.05 * 0.02 V obtained for D. demifzirricans Norway cytochrome cssJ [2 I]. In fact it appears from AEp = Epa - Epc values that cytochrome css3 is not a strictly reversible system. The rate constant for electron transfer can be estimated from the peak potential separations at increasing scan rates [22]. A k, value of about 1 X 10v3 cm s-’ has been obtained by using the Nicholson treatment, which means that the electrode process can be defined as a quasi-reversible one 1231 (see Table 1). way
Effect of pH Differential pulse and cyclic voltammetry peaks with increasing pH are found to be unchanged up to a pH of about 10, then to decrease above this value.. The simultaneous alteration in the absorbance of the 690 nm band in the oxidized form is shown in Fig. 3, indicating- that the structure of cytochrome c,,, is modified; a decrease is also observed for the absorbance of the 529 nm absorption band in the oxidized form. Sharp drops are observed above pH 10 in Fig. 3 on .both absorbance; pH curves. The quenching of the 690 nm band is consistent with the disruption of the iron-methionine bond. It seems that this quenching is also associated with the decrease of the 529 nm band. It may be assumed that a new “alkalke” form of cytochrome css3 app ears: a pK value of 10.9 -C 0.1 for the transition between both, cytochrome cfrs3 forms can be determined from the midpoint pH of both absorbance; -pH curves. This pK value is noticeably higher ‘than. the.pK_ value.(9;1) .&$&ted :ordinarily for the methionine to lysine bonded heme :iransition- of --ho&e-heart:. .
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Fig. 3. Effect of ;H-on thi absorbance of the 6&I nm-band (1. left-hand scale) and of the 529 nm band (2. right-hand scale) of 150 gM cytocbrome css3 in (0.05 M sodium phosphate+O.OS M sodium borate) buffer.
cytochrome c [ 161.Con&-y ‘to the existence: of an. ‘electroactiie “alkaline” cyto-chrome c form [6], no.other peak has been detected in Qoltammetry experiments-on cytochrome css3by increasing the pH in the potential -range investigated. DISCt.JSSION : ~. -
:
Several -c-type i3ytochromes a& present in- Desulfooibrio species. The typical cytochrome of the genus ~Destilfmibrio..is~the multiple-heme &&chrome c,, but D. uulg&s cyto@rome c5& is a partic&rly iritq&ing h&noprote&: it iS a single-heme pro&i of Imoderate molecular weight,- like -mitochondrial’ cytochrome c, -with the sax& typk<.of _h&mk’:and k&nt attachmejnt of he&e to -.the proteinic chain. CytoChro~tic;,~ has b&n comp+d to’other cytochrom+ c by Dickerson et al. [24], and:@ of $e hemofiioteins’ of this f&nily seerp to be tilosel$ related by evolutionary hqm&gy_:-.
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In&&i, -in. ihi. present.work,: it ,.hti bEti &ablished from differentjal ptilse and dydlic i&m&e&y .$t: the’ go@ :eiectrode in the : presence of 4,4’-bipyridine as activat&ag&t that th& v$ie of.t+&dqx-po@iial EA is very close to 0.02V::A more.‘.k&ativ& -.v$tik’ &.s- b&n dbta@d Iby- Ya& ,.[lO] for D. ~&l&zris~Miyaza&i Ai --pd&&i .&it- ‘69 ~:Stell$a&n cy@chiofle cSs3frtim_,e@ilib~ti~~~ ‘+Fa$u&ke&. [25]; -the-redox potentiai-~~~~heiiie:ih~~d.~~e &+k ~qd:&@rk&&iive. %l!i&:its ~,l . __I-..._ .-
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percentage exposure .to solvent increases. In the case of cytochrome c553, by using the relationship E,’ (V) = -0.015P + 0.345, a percentage exposure P of 22% could’ be predicted. This partial accessibility to the heme could partly explain that, even in the absence of the activating agent 4,4’-bipyridine, a slight current response can. be detected at the gold electrode by differential pulse and cyclic voltammetry. At the mercury electrode, a peak is observed at Et, = -0.20 V (vs. Ag/AgCl, i.e. E, = -0.02 V vs. normal hydrogen electrode), which correponds to a cathodic overpotential of ca. 40 mV. In fact. the exposure of the active centre is not the only factor which governs the kinetics of electronic exchanges at electrodes. A reaction scheme for the electrontransfer reactions of metallo-proteins at electrodes has been proposed by Albery et al. [26]. where a considerable binding energy for the preceding adsorption step seems to be essential. The binding of proteins to the electrode surface has been compared with the bindings to its physiological partners. From kinetic studies on several cytochromes c, Errede and Kamen [27] have shown that the strength of binding is a dominant parameter in reactivity. It appears that this factor must be taken into account to draw a parailel between the behavior on the electrode and the physiological process. The fact that 4,4’-bipyridine acts as an activator agent in the case of both cytochrome c and cytochrome cssj suggests the existence of similar binding interactions and supports the assumption of a prerequisite adsorption step. A comparison between cytochrome cgj3 and horse-heart cytochrome c is of special interest_ Both cytochromes are monohemic, with respective molecular weights .of 9,100 and 13,000. In both cases the axial ligands of the iron atom are the same, i.e. methionine and histidine residues. However, their redox potentials are separated by about 240 mV. The most significant differences have to be sought in sequences and structures. It has been noted by Cookson et al. [9] that for D. ordgaris cytochrome cSS3 there is only limited evidence for structural homology with other c-type cytochromes. The only aromatic amino acids in the cytochrome c,,, sequence are tyrosine residues, and so.me of these tyrosines are far from the heme; it has been suggested that the heme environment is less hydrophobic. The results of our study tend to support the conclusion that the heme environments of horse-heart cytochrome c and D. odgaris cytochrome cSs3 are rather different. Another striking feature is the strong resistance to the effect of increasing BH on the transition between the “neutral” (= iron-methionine bonded heme) and “alkaline” (= iron-methionine unbonded heme) forms. This result suggests that the iron-methionine bond is relatively strong in the case of cytochrome- cssj; and corroborates the fact that a stabilization of Fe(W) state and hence a lower redox potential may be observed with this cytochrome [9]; the ability of methionine to donate electrons is dependent on the protein structure. REFERENCES 1 P. Bianco and J. Haladjian, Biochim. Biophys. Acta, 545 (1979) 86. 2 P. Bianco. G. Fauque and J. Haladjian. Bioelectrochem. Bioenerg.. 6 (1979) 385.
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-4 P.-B&&~&i J; H&&j&n. B&&c&he~n.Bi&snerg.. 8.(1981)-239. .~ :.’ 5. J: Haiadjian, P. B&i& &td P.A.‘Serre, I.- Eiectroaqal. Chem.. 106 (1980) 397., ! --6 P.A- &&;_J::Haladjian @td -P. Bianco, J:Electr~tal. Chem., i22.(i98i) 327. .- .-7. J. Le .Gaii and M. B&&hi-Heriaud;~iri~~K. 0kunuki;M.D. Kamen and J. Sckuzu (Ed%), Structure and : &n&k~f Cy&tm~&$ Ut&$ty of Tokyo Press and University-Park Press, -1968, p. 467, 8 M~B&scM~and_J. Lit Gaili Biochim.=Biophys; Acta, 271 (1972) 48. -9 D-J: Cooksort,~G.R. Moore; RC. Pitt. R.J.P.~_WiBiams. I.D. Campbell. R.P. Ambler. M. Bruschi and J. Le.GaB. Eur. J. B&hem.? 83 (1978) 261:. 10 T. Y&i B&him:_ Biophys.. Aeta., 548 (1979) 96. 1i K. Nii. T:Yagi; H. Inokuchi and KKimura. J. A& Chem. Sot., 101 (1979) 3335. 12 W.F. Sokoi, D.H. Evans, K. N$i and T. Yagi, .T. Elcctroauai. Chem., IO8 (1980) 107. i3 W.R. Heirreman, BJ. Norris and J.F. Goeiz, Anal. Chem., 47 ( 1975) 79. 14 M.f. Eddowes and H.A.G. Hill, J. Am.~Chem. Sot.. 101 (1979) 4461. I5 M.J. Eddowes, H.A.O. HiBand K. Uosaki, Bioeiectrochem. Bioenerg.. 7 (1980) 527. 16 R Dick&on and R. -Timkovich in P.D. Boyer (Ed.), The Enzymes, Vol. 1 I, Academic Press. New York; 1975, p. 397. 17 H. Hasumi, Biochim. Biophys. Acta. 626 (1980) 265. IS F. Scheiler, Bioclectrochem. Bioenerg., 4 (1977) 490. 19 B.A..Kuznetsov, G.P. Shumakovich and N.M. Mestechkina, Bioeiectrochem. Bioenerg.. 4 (1977) 512. 20 R. Bates, Determination of pH. Wiley. New York. 1964. p. 123. 21 G. Fauque, M. Brusbhi and J. Le Gail, Biocherrt. Biophys. Re.s. Commun., 86 (1979) 1020. 22 RS. Nicholson, Anai. Chem, 37 (1965) 1351. 23 A.M. Bond, Modern Polarographic Methods in Analytical Chemistry, Marcel Dekker, New York and Basic;i980, p_ 24. 24 R_E_ Dickerson, R Timkovich and RJ. Almassy. J. Mol. Bioi., 100 (1976) 473. 25 E. Steiiwagen; Nature, 275 (1978) 73. 26 W.J. Aibery, M.J. Eddowes, H.A.0, Hill and A.R. Hiiiman, J. Am. Chem. Sot.. IO3 (1981) 3904. 27 B..Errede and M.D. Kamen, Biochemistry, 17 (1978) 1015.