Spectroscopic study of the compound ES and the oxoferryl compound II states of cytochrome c peroxidase: comparison with the compound II of horseradish peroxidase

Inorganica Chimica

Actr 275-276

( 1998) 250-255

Spectroscopic study of the compound ES and the oxoferryl compound II states of cytochrome c peroxidase: comparison with the compound II of horseradish peroxidase ’ Alycen E. Pond II,Grmt S. Bruce ‘, Ann M. English b, Masanori Sono ‘, John H. Dawmn ulOe* * Depurtmenr of Chemistry and Binchemistr?: Univemity c$Svuth Curolrrrcc.Colwnbiu, SC 29208, USA ’ Lkpment of Chemistt?: and Biochomistty, Concordia Univemity, Montreal, H3G IMN, Cnnc~ri~t ’ School ofhfedicine, University of South Carolina, Cdumhio. SC 29.?@8. USA Received 5 June 1997: accepted 2 September

1997

- WC we rcpot~l for the oxoferryl compound II state of cytochrome f de oxidation of ferrous CcP. in comparison with compound ES fun oxofcrryl I (an oxofertyl heme) of horseradish pcmxidusr: ( HRP-Ii 1. D&&d spectral I3 does not significantly perturb the electronic anvimnmt?ut of the hcme us stutesnf’CcP aresimilar. ro~scopy.Thus, the spectra of the compounds 1 and II ( oxakrryl) aher hand, contaiils un oxafcrryl hcme ~upkd to R porphyrin r-cation rudicul. As u n have quite dintinctivc spectra, The rimilority nf the spectral propertics of the twu uctivu Iween Ihe spectr&l propertics of the two active high vulcnt q%cias of NRP. clrrttrly indicntos r0 IWM the importwe 6f the pro&in slruclurc rrurruunding the hems group in dekrmining the ap~tnl propflea d the active q~ics. Blsigvler Seicaeo SLAtiAll i@tttr reserved,

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c proxidase (CcP) ’ is u solubk 34 kx;)u the mitochondrirtl intcrmembrune srribtion of femytazhrome c by M * =+2Cyt i?(i%’ + ) + 2N,6

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s&emutiaiily &own in Fig. I. the ration proceeds a multi-step mechanism,In the Wrst step, the resting tateoft%P ia oxidizedby I&t& to fQrtn compamdES

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1aThis spaAs is two ~)xidi~in~equivulentsubow state,containingun 0xaferryl~Cl md I protein rudicul, and is equivalent la compoundI (un oxoferryl heme with H porphyrin Iration radical) of other proxidassa such us WRP. The Iacutianof the protein rudical has beenthe focus of numemuastudies [ 2-S] with rhe conSCttiU ing the radicalon the indolering afTp 191. CcPfiS is ueedto compoundII (CeP’II ) by one molecules of fermcytoshrrrme c, Mowed by reduction of CcP’ll by u secondmcrlccult:of ~~~~~yt~h~ui~ c buck to the resting ferric strac.TWO frrrms of CcY’kl wete idcntilIr?dby Coulson et al. (41. iYcP’I1,. (IV. Trp) with un oxofcrryl hcmc,and CcP’&tR f HI. Trp’” 1 with 3 faric hemr und a rudicul on the protein (presumablyon Trp191). both of which are in equilibrium as u result 0T intaaneleculiirelectrontransfer between the protein radicalsite and the hemt iron. One method by which the axeferry1 iron can be formed without the concomitantformation of the protein free radial is the hydrogenperaide oxidationof the ferrous stateof CcP IV. The tesuhing form of CcP’II. r&a-red to hereafter us

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Chimlca Acn 275-276

2.2, Pmpatwtion of mnpk CcP crysuds [20] were dissolved in 50 mM potassium phosphate,pH 7.4, and then dialyzed against the samebuffer. The dialyzed solution (A,,IA,, = 1.30) was then concentrated to 2-3 mM. CcP concentrationswere determinedspecttophototnetrically using a miilimohtr extinction coefficient at 40%nm of 98 mM - ’cm- ’for the native ferric enzyme at pH6-7 1211. The buffer system usedfor the photoreductionexperiment consistedof 49% ethylene glycoi, 49% 100 mM potassium tc (pH7.4),2%ioopropanol,O.O1% (voi./voi.) acenone, and O,i nM cat&~. The isopropanol was retluxed with solid stannouschloride to remove peroxides [ 22 ] and then storedunder a nitrogen atmospherein the dark. was dialyzed cxtmsiveiy against deionized water 20 mM potassiumphosphatebuffer, pH 7.4 at 4°C The ct~lase had an Aa,3/Az7hvalue of 0.9 or greater. After the addition of cataiase, the buffer mixture was allowed to stir for at least 30 min to remove any residual tracesoihydm roxide. IP most critical aspect of generating homogeneousferthe photoreduction technique was to ensure

passed through a chromous in solution w91sthen

(1998)

250-255

were measuredat 1.4 1 T with a JASCO J5OO-A spectropolarimete: equipped with a JASCO MCD-IB electromagnet and interfaced with a Gateway 2000 4DX2-66V PC through a JASCO IF-500-2 interface unit. Spectral measurements were performed at 4 or - 30°C as indicated. Data acquisition and manipulation has been described elsewhere [ 251. UVVis absorption spectra were recorded before and after the MCD measurementsto verify sample integrity. EPR measurements were obtained at 77 K using a Varian E-9 Century Series spectrometer equipped with an E-102 microwave bridge.

3. Results und discusiun Fe( 111)cytocbrome c peroxidasc was readily and quantitatively photoreducedto form a speciesthat had UV-Vis and MCD [ 241 spectraconsistentwith a fully reduced, five-coordinate, high spin ferrohemoprotein (Fig. 2(a) ) 1261. The ferrous complex was stable at both 4 and - 30°C for more than 40 min. Upon addition of one molar equivalentof hydrogen peroxide to 2 10 p.M ferrous enzyme, its UV-Vis absorption spectrum changed to that of another species with the series of transient spectra exhibiting isobestic points at u 545 ;md h 600 nm when monitored at 2 min intervals (Fig. 2(b) ). This relatively slow conversion rate may not be unreasonablydifferent from that previously reported for the conversion of deoxyferrous HWP to HRP compound II by

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CCP’II, Was complete in uppmxit ‘h? sample was Irunsfcrred to the

spectrumwas very similar fo thut CcP==ES ( vidr?ini’m), isitting the presenceof un oxolhrryi hems. and, possibly, a protein-baxed free radical, possible decomposi0on of the hyd~~~~n peroxide by the culaiaset(0. I nM I pmsont system was n~~ii~ibt~ under the conditions in the bu ausc the photoreductionstep also producedferemployed rous cutaiasls(B.S. Bruce und J.H. Dawson, unpublished results) which wlas pFccsum&iythen oxidized by hydrogen peroxide to the axofe~i form ( i.e., compound tl ), which is a c~~t~~lyt~c~lly inactive form of eataiase. Due to the difflcuity of distiuguishing CcP-ES ondCcP’IIII using absorption spectroscopy, EPR spectra of both were measuredunder the same conditions ( Fig. 3 1. C&-ES has ;I strong signal at t~ppmximUeiy 8 = 2.0 which urises from the amino acid-based free radical [2-5 1, The CcP species thus prepared in this work by the addition of I molar equivalent of M&I2 flow CcP (Fig. 3, inset 1 essentially lacks this or less af the signal intensity of CcP-ES in the signal I prepanrtionnused). The week EPR signal seen for the CcP species, coupled with the similarity of its absorbancespecttum tn that crfCcP-ES, supports its assignment as an oxoferry1 heme, CcP‘II,: ( IV. Trp. Fig. I ). The classification of the species as an oxofcrryi heme was suppolted by earlier combined EPR and spctmphotometric studies a’lricb

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Fig. 3. EPR spectra of CcP’II+ ;md CcP-ES. Main spectra. CcP-ES: 300 )LM CcP in 49% ethylene glycol. 49% I00 mM potassiumphosphate( pH 7.0). 2%isopropmol. 0.01% rcetophenotte.0.1 nM catulrse buffer. 3 mm intcmul diumcter quattz EPR tube. SC~II mnpe: 5000 G; field set: 2500 G; time constitnt:0.032 s; scnn time: 4 min; modulation amplitude: 10 G: tnodututionfrequency: 100 kHa; receiver gain: 500: temperuture: 77 K: microwuvepower: IO mW; microwave frequency:9.06GHz. htset.CcP’II, : 300 (LM CCP. The solvent system. the insttumentparutneters.and the other conditionswere the same in both cases.The inset was offset for clarity. Both signalshad g values of 1.0. Note that the sign of the EPR spcc~m is invetted in the ligure.

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ilar, although the a+-and P-peaks for CcP’II,, have similar intensities (d/3 = 1.OJ). Conlparison with HRP-II (dotted line) emphasizes the similarity between the two high valent species of CcP, and their variation in both position and intensity from that of HRP-II. In agreement with the likeness of the absorption spectra, the MCD spectra of the IWO CcP species ‘II%also very similar (Fig. 4( a 1) with CcP-ES once again bcmg slightly more intense. The MCB spectra of CcPES and CcP’ll,. differ from HRP-II in the visible region in IhaI they have an intense derivative-shaped feature wirh a cross-over at 562 nm which COIIIP(\SISwith iI much less intense fenturc for HRP. The similarity brlwcetr CcP-ES andCcP’ll,, was expected since the protein-based free radical has been shown tn oaly slightly pcrturh the spcstral propenies CcP 1‘71, but the divergence from the spectra of HRP-II is noticeable. A possible reason for this MCD specrral difference WY be further considered below. Numerous studies have focused WI the complexity of Ihe catalytic cycle CcP. specifically on the one-eleeWonreduc” lion steps from CcP-ES to CcP-II, and from CcP-ll to the resting ferric state [ I 1, Pre-steady stale kinetics of the reduction of the CcP-ES with ferrocyanide I291 suggested that the free radical site is reduced approximately live-fold faster than the axoferryl heme. However, results of an equilibrium reductive titration of CcP-ES with ferrocyanidc at acidic pH vdues ( <6) determined that with every aliquot of fWfOCy;lui& there is as much free radical as ferry1 hcme reduced 16 I. These results suggest lhet the hcme and the free radical sites exchange oxidizing equivalents, in particular, that the radical site once reduced can be reoxidized. &her intra- or intermolecularly, by Ihe ferry1 heme. Recently. it has been determiued that ferrocyauide may utilize a number of differellt pathwaysfor reduction of the oxidized enzyme I 301, and lndy not be the best probe of what is occurring during the physiological turnover of CcP by ferrocytochrome t’. In this

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fcvealed an equilibrium between the (111. Trp” ) and (IV. Trp) forms ofCcP-11 that is dependent on pH 16,281. Al pH 7.4 in the present study, the equilibrium favors nearly camplete formation of the oxoferryl heme species [ 6,28 1. Hettcc, a closer comparison ofCcP’11,. to CcP-ES and the oxoferryl species HRP-II was undertaken. The electronic absorption spectra of CcP-ES, CcP’II,.. and HRP-II are presented in Fig. 4(b). In agreement with the results reported by Hoffman and co-workers 17 1, the spectrum of CcP-ES (dashed line) has fenlures that are more intense (by w 15%) than those ofCcP’III;. both in the visible and Sore1 regions and has an a-peak ( 560 mn ) hat is somewhat more intense than its P_peuk ( 530 nm) ( a//3 = I. I3 1. The peak positions and general spectra of CcP’II,: ( solid line. Fig. 4(b) ) and of CcP-ES (dashed line, Fig. 4(b) ) we sirn-

AX. Pod et ul. /Inorganica ChimicaActu 275-276 (1998) 250-255

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rapid intracomplex electron transfer occurring only at the high-affinity site identified in the crystal structure 133,341. Binding of yeastcytochrome c to the low affinity-site appears to lower the affinity of yeastcytochromec for the high-affinity

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respect,a study by Ortiz de Mr~atellsnoand ~-workers [ 3 I 1 of the ~~~~~ a~tivitios of CCP mastiwt ftd hems has shown that ferrocyenideand fe t* are exidi at different CcP sites. The nrtuml substratefor CcP, cytochrome c. also demonstratesPreferential reduction betwc~n the two sites which is nt on ionic @ength, and the source nfcytochrome c * diffemrces may 1%the resu the manner in which the substrate interacts with CcP. tier and Kraut I 32 1 detemi through X-ray crystrrllogruphythat horse cytochmme c und yetrstcytochromet* bind t

sites are available for ymt cytochrome c nity by appximta2ly IO00 fold with the

site at low ionic strength. This possibly results from some type of conformationai variation within the protein which

could effect the mode of intracomplex electron transfer r33.341. Stopped flow experiments with native horse heart cytochrome c and yeast cytochromec showed that the radicalsite was preferentiallyreduced at high ionic strength [ 35 I, while the oxofenyl site was preferentially reduced at low ionic strength [ 361. When ruthenium derivatives of horse cytochrome c and yeast cytochromec were employed at low ionic strength, kinetic data supportedthe reduction of the radical site initially, followed by reduction of the oxoferryl heme [ 28,311. While the mechanisms presented by these studies disagreed on the initial site of reduction, both predicted an equilibrium between the two sites,and therefore, intramolecular transfer between the sites. This intramolecular transfer within the catalytic cycle of CcP is shown in Pig. 1. With this scheme in mind, the formation of some percentage of the second form of CcP-II halving the ferric heme iron and the radical on the tryptophanwas expected.The ptesenceofferric hcme would alter the electronic absorptionand MCD spectral propertiesof CcP’II,:,and could accountfor the spectraldeviation from HRP-II. However, the lack of a EPR signal for CcP’Il,, indicates that there is little to no intramolecularelectron tmnsfer occurring tween the heme iron and the protein radical site. Therefore, c oxoferryl heme in CcP’Il,, is unable to oxidize the radical site, nnd intramolecular transfer cannotbc the explanation for the spectraldiffennceshetween CcP’II,,and HRP-11. The differencesin the electronic ahsorption and MC WI1olCcP’II,. versus HRP-11 are probably indictrtiveot ens in the protein conformation surroundand its interaction with the active site. The catalytic cycle proposedby Hoffman and co-workers 17 1, which predicts that the protein conformation of the radical site may alter the oxidiznbility of the site, could account for the apparentinhibition of intramolecular electron transfer. The lack of radical formation for CcP’IIi; supports the assumptionof two protein confcnrmationsfor the mdical site which have varying reduction potentials. The evidence that the oxoferryl heme in CcP’IIV does not noticeably oxidize the mdical site, es ially at pH valuesabove 6, could indicate that the conformation of the site in CcP’IIij is in an unfavorable state, i.e., one with a high reduction potential. The similaritiesdthe electronic absorptionand MCD spectraofCcPRS and CcP’& indicate that neither the radical itself nor the protein conformation of the site appearsto affect the spectral propertiesof the protein. In conclusion, it would appear that the protein-based free radical found in CcP-I!!5 does not significantly perturb the electronic environment of the heme as measured by absorp tion spectroscopyand MCD spectroscopy.This linding is in contrast with that for HRP which has the second oxidizing

equivalent in the form of a porphyrin n-cation in its compound I state and is well known to have quite distinctive spectra for HRP-I and HRP-II.The similarity of the spectral properties of the two high valent active species of CcP. and their apparent variation from the analogous forms of HRP, indicates that the protein structure surrounding the heme group may play an important role in the spectral properties of the active species. The apparent lack of intramolecular electron transferbetween the two sites of CcP reportedherein is consistent with the pH-dependentequilibrium between the ferric heme/protein radical and oxoferryl heme states, the latterbeing favored at pH > 6 [ 6,281. These results also support the proposed existence of two varying conformationsfor the radical site [ 71, which differ in their reduction potentials und ability to stabilize the radical.However, thesedifferences in the protein structureof CcP-ES versus CcP’Il,; appearto be undetectable by the methods employed here. These results furtherillustrate the complexity of the catalytic cycle of CcP which is dependent not only on the source of electrons, but the conformation of the protein for optimal activity.

Acknowledgements

We thank Drs Edmund Svastits and John J. Rux for assembling the MCD softwarc and JenniferCheek and MarkRoach for helpful discussions. Support provided by NIH GM26730 (J.H.D.) and the NSBRC ( A.M.E. ). The electromagnet for the CD spcctrophotometer was purchased through a grant from Research Corporation.

J.M. Muttro, LA. Fishcl. J.T. Hurzurd, T.E. Meyer. G. Tollin, M.A. Curunovich und J. Knut. Biochemistry. 27 ( 1988) 6243. 141 3.6. l%ttvm, L.B. Vitello. J.M. Muuro und J. Kruut. Biochcmislry, 2X (1989) 7992. [ 51M. Sivnruju, D.B. Goodin. M. Smi!h und B.M. HofTtau~~, Science, 245 (IYX9I 73x. 161 A.P.W. Cctulso~t. 1.8. Ermtm und T. Yonctuni. J. Biol. Chem.. 246 (1971) 917.

I71 P.S. HO, B.M. Hoffman, C.H. Ksng and E. Margoliash. J. Biol. Chem.. 258 ( 1983) 4346-1363. I81 B. Wtud and C.K. Chimp. Photochem.Phaiohiol.. 35 ( 1982)757. 191 J.H.DuwsonandD.M.Dooley,inA.B.P.Ltver~~dH.B.Gr~yteds.). Imn Porphyrins, Part III. VCH. New York. 1989, pp. l-135. [ IO] L.E. Vickery. T. Nozuwa and K. Sauer,J. Am. Chem. Sot.. 98 I 1976 J 3. [ II] S. Suzuki, T. Yoshimum. A. N&&u-u, H. Iwus&. S. Shidumand T. Mutsubara,Inorg. C&m.. 26 ( 1987) 1006. II21 D. Meyers and G. Palmer. J. Biol. Chem., 260 ( 1985) 3887.

[ 131 R. Evangel&t-Kirkup. M. Crisanti. T.L. Poulos andT.G. Spiro. FEBS Lat., I90 (1985) 221.

[ 141S. Hashimoto. J. Tenoka, T. Inabushi,T. Yonetani and T. Kitagawa. J. Biol. Chem.. 261 (1986) 1I 110. [ IS] S. Dasgupts.D.L. Rousseau.H. Anni and T. Yonetuni. J. Biol. Chem.. 264 ( 1989) 654. [ 161 P. Hildcbnndt, A.M. English and G. Smulcvich. Biochemistry. 31

t 1992b2384. I I?] S.L. Edwrrds. N.H. Xuong, R.C. Ihunlin irndJ. Kruut. Btochcmistry, 26 (1987) 1503. I81 D. Dolphin, A. Formun. DC. Borg. J. Fajer und R.N. Felton, Proc. NatI. Acad. Sci. U.S.A.. 68 (1971) 614. 191 J.E. Roberts. B.M. Hoffman. R. Ruttcr and L.P. Hager. J. Am. Chem. Sot.. 103 ( 1981) 7654. I201 A.M. English. M. Laberge and M. Walsh. Inorg. Chun. Acta. I23 ( 19X6) 113. 121 I T. Yonetani and H. Anni. J. Biol. Chcm.. 262 ( 1987 J 9547. 1221 D.D. Perrin and W.L.F. Armago. Puritication of LaboruIory Chemicals. Pergrmon Press,New York, 3rd edn., 1988, p. 207. I23 I A.J. Gordon und R.A. Ford, The Chemist’s Compunion, o Himdbook ofPmctic;ll Dst;~.Tcchniyuesund Rcfereuces,Wiley, New York, 1972, p. 438. 1241 G.S. Bruce. M.Sc. Thesis, Univcrsily of South Carolinn. Columhla. 1986. I 15I A.M. Huff, C.K. Chang. D.K. Coopcr. K.M. Smith imtlJ.H.Duwson, huq% Chcm,, 32 ( I993 I IShO. I261 HA. Wiltenhcrg, L. KIIIII~. J.B. Wittenberg. WE Wlwhmp ml J. Peisuch.J. Biol. CIIWII.,243 ( lO6H) IX63 127I R.W. Nobls uad Q.l-1,G~bnon.J. 131trl. Cltm.. 255 ( 11140) ?dW I ?H] R,*Q. Liu. M.A. Miller. S.W. Nun. S. Hnhm, I_. (irmn. S. lhMo~t.J. Kmu1. N. Durhnm und P. Mills& Biochenuslry. 33 ( I994 I HbYti I ?OI 1I.C’.Jordi IIII~ 1.13.Ermun, Btochemihtry. I1 ( IO74 b3734. I30) SK. Wilcox, GM. Jenaca. M.M. Fi~,!geruld,&.E. M&cc nnd l&IS Goodin. Biochemistry, 35 ( 1996) 48%. ( 311C1.D.Rpillis, BP. Sishtu, A.Ci. Muuk r~mlP.R. 0rtir dc Montell~uro, J. Bird. Chum., 266 ( 1991 ) 193.14, I 32 I W. Polletier und J. Knut, Science, 2Sn ( I992 1 17411. [ 3.71M.A. Miller, Biochemistry. 35 ( 1996) 15791 1341 H. Mei. K. Wang, S. McKee, X. Wung. J.L. Woldner, G.J. Piclak, B. Durham ru~tl F. Millet& Eiochcmistry, 35 ( 1996) 15800. 1351 S. Huhm. B. Durham und F. Millcct. 1. Am. Chcm. Sot.. I IS ( 1993 I 3372. [ 361M.R. Nuevn,H.-H. Chu. L. Vitcllo und J.E. Emtun. J. Am Cbcm sot., I I5 ( 1093) 5873. I37 I 1,.C&n, S. Iluttnt.8. Durhum undF. Millctt. Biochcmihtry.30 ( 1991 ) 0450.