5
Proc. Nati. Acad. Sci. USA Vol. 84, pp. 6687-6691, October 1987 Biochemistry Spectroscopic analysis of the cytochrome c oxidase-cytochrome c complex: Circular dichroism and magnetic circular dichroism measurements reveal change of cytochrome c heme geometry imposed by complex formation (electron transfer) CHRISTOPH WEBER*, BRUNO MICHEL, AND HANS RUDOLF BOSSHARD Biochemisches Institut der Universitdt, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland Communicated by E. Margoliash, June 4, 1987 (received for review March 11, 1987) ABSTRACT Binding of cytochrome c to cytochrome c oxidase induces a conformational change in both proteins as well as a change of the electronic structure of the heme of cytochrome c, indicating an altered heme c-protein interac- tion. This follows from the observation that the induced circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the oxidase-cytochrome c complex in the Soret region differ from the summed spectra of oxidase plus cytochrome c. Spectral changes occur in the complex composed of either the two ferric or the two ferrous hemoproteins. The difference CD and MCD signals saturate at a ratio of 1 heme c per heme aa3. The difference spectra are specific to the cognate complex. The results are interpreted to reflect a direct relationship between the recognition/binding step and the electron-transfer reaction. The conformational rearrangement induced in cytochrome c by cytochrome c oxidase consists of a structural rearrangement of the heme environment and pos- sibly a change of the geometry of the heme iron-methionine-80 sulfur axial bond. This rearrangement may decrease the reorganizational free energy of electron transfer by adjusting the heme c geometry to a state between that of ferni- and ferrocytochrome c. Mitochondrial cytochrome c catalyzes the electron transfer from ubiquinol-cytochrome c oxidoreductase to cytochrome c oxidase (cytochrome aa3; EC 1.9.3.1) in the final segment of the respiratory chain. Formally, the reaction can be divided into a recognition/binding step and an electron- transfer step. There exists a large body of information about the structural elements of cytochrome c involved in the recognition/binding step. The electron-transfer interaction domain of cytochrome c recognized by cytochrome c oxidase and other electron acceptors as well as donors encompasses the top front, most of the exposed heme edge, and part of the left side of the molecule (1-5). Correlating this structural information with a plausible mechanism of electron transfer turned out to be difficult. The contribution of the specific recognition/binding step to the efficiency of electron transfer still has to be clarified. On the basis of theoretical considerations it is expected that prior to the electron transfer the heme cofactor of cytochrome c adopts a coordination environment that is half-way between the environments of oxidized and reduced heme ct (6, 7). This follows from the Frank-Condon princi- ple, which predicts that the nuclei of the hemes do not have time to change position and momentum during actual electron transfer. However, a direct correlation between the predicted adjustment and the recognition/binding process remains to be shown. Spectroscopic differences in circular dichroism (CD) and magnetic circular dichroism (MCD) indicate changes of the protein matrix after the electronic change Fe(II) = Fe(III) (reviews in refs. 8 and 9). From a comparison of the crystal structures of reduced and oxidized tuna cytochrome c some of these changes could be attributed to a small concerted movement of side chains and backbone on the methionine-80 side of the heme (10). If binding to a macromolecular redox partner were to induce related changes, the electron transfer might be facilitated. Possible evidence for a conformational rearrangement was obtained by our previous observation of a small perturbation of the heme c spectrum when cytochrome c binds to the oxidase (11). The spectral change must be a reflection of a changing heme environment, since a direct heme-heme interaction can be ruled out (ref. 12 and references cited therein). Here we report changes in CD and MCD spectra in the Soret region when cytochrome c and oxidase form a tight 1:1 complex. EXPERIMENTAL PROCEDURES Cytochrome c (horse) was Sigma type VI and was used without further purification. Porphyrin-cytochrome c (13) and apocytochrome c (14) were prepared as described. Phosvitin (Sigma), poly(L-glutamate) (Fluka, average Mr = 32,500), and spermine (Fluka) were used as supplied. Beef heart cytochrome c oxidase was prepared according to Hartzell and Beinert (15). The enzyme had 9 nmol of heme a per mg of protein and 18 mol of phosphorus per mol of heme aa3. A d/A422 was 1.33 and A42x/AYID was 2.55. The maxi- mum turnover number with ferrocytochrome c was 170 s at 20'C in 10 mM Tris.HCI/15 mM NaCl/0.05% dodecyl maltoside, pH 7.4. This buffer was used for all spectroscopic measurements. The enzyme was brought into this buffer by ion-exchange chromatography (16). Concentrations of heme c and heme aa3 were determined as before (11). CD and MCD spectra were recorded at 25 ±+ 1C on a JASCO (Tokyo) spectropolarimeter (model 500C). MCD spectra were recorded with a magnetic field of 1.5 tesla (T), parallel and antiparallel to the light beam. The scan rate was 20 nm/min and the time constant 1 or 2 s; seven spectra were accumulated at both orientations of the magnetic field. The Abbreviations: CD, circular dichroism; MCD, magnetic CD. *Present address: Institut fur Molekularbiologie und Biophysik, ETH-Honggerberg, CH-8093 Zurich, Switzerland. t"Heme c" indicates the heme of cytochrome c. "Heme a" and "heme a3" indicate the two spectroscopically different but chem- ically identical hemes of cytochrome c oxidase. "Heme aa3" indicates the sum of heme a and heme a3. 6687 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Page 1: Spectroscopic Circular dichroism - Proceedings of the ... · PDF fileProc. Nati. Acad. Sci. USA Vol. 84, pp. 6687-6691, October 1987 Biochemistry Spectroscopic analysis ofthe cytochromec

Proc. Nati. Acad. Sci. USAVol. 84, pp. 6687-6691, October 1987Biochemistry

Spectroscopic analysis of the cytochrome c oxidase-cytochrome ccomplex: Circular dichroism and magnetic circular dichroismmeasurements reveal change of cytochrome c hemegeometry imposed by complex formation

(electron transfer)

CHRISTOPH WEBER*, BRUNO MICHEL, AND HANS RUDOLF BOSSHARDBiochemisches Institut der Universitdt, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

Communicated by E. Margoliash, June 4, 1987 (received for review March 11, 1987)

ABSTRACT Binding of cytochrome c to cytochrome coxidase induces a conformational change in both proteins aswell as a change of the electronic structure of the heme ofcytochrome c, indicating an altered heme c-protein interac-tion. This follows from the observation that the inducedcircular dichroism (CD) and magnetic circular dichroism(MCD) spectra of the oxidase-cytochrome c complex in theSoret region differ from the summed spectra of oxidase pluscytochrome c. Spectral changes occur in the complex composedof either the two ferric or the two ferrous hemoproteins. Thedifference CD and MCD signals saturate at a ratio of 1 hemec per heme aa3. The difference spectra are specific to thecognate complex. The results are interpreted to reflect a directrelationship between the recognition/binding step and theelectron-transfer reaction. The conformational rearrangementinduced in cytochrome c by cytochrome c oxidase consists of astructural rearrangement of the heme environment and pos-sibly a change of the geometry of the heme iron-methionine-80sulfur axial bond. This rearrangement may decrease thereorganizational free energy of electron transfer by adjustingthe heme c geometry to a state between that of ferni- andferrocytochrome c.

Mitochondrial cytochrome c catalyzes the electron transferfrom ubiquinol-cytochrome c oxidoreductase to cytochromec oxidase (cytochrome aa3; EC 1.9.3.1) in the final segmentof the respiratory chain. Formally, the reaction can bedivided into a recognition/binding step and an electron-transfer step. There exists a large body of information aboutthe structural elements of cytochrome c involved in therecognition/binding step. The electron-transfer interactiondomain ofcytochrome c recognized by cytochrome c oxidaseand other electron acceptors as well as donors encompassesthe top front, most of the exposed heme edge, and part of theleft side of the molecule (1-5). Correlating this structuralinformation with a plausible mechanism of electron transferturned out to be difficult. The contribution of the specificrecognition/binding step to the efficiency ofelectron transferstill has to be clarified.On the basis of theoretical considerations it is expected

that prior to the electron transfer the heme cofactor ofcytochrome c adopts a coordination environment that ishalf-way between the environments of oxidized and reducedheme ct (6, 7). This follows from the Frank-Condon princi-ple, which predicts that the nuclei of the hemes do not havetime to change position and momentum during actual electrontransfer. However, a direct correlation between the predicted

adjustment and the recognition/binding process remains tobe shown.

Spectroscopic differences in circular dichroism (CD) andmagnetic circular dichroism (MCD) indicate changes of theprotein matrix after the electronic change Fe(II) = Fe(III)(reviews in refs. 8 and 9). From a comparison of the crystalstructures of reduced and oxidized tuna cytochrome c someof these changes could be attributed to a small concertedmovement of side chains and backbone on the methionine-80side of the heme (10). If binding to a macromolecular redoxpartner were to induce related changes, the electron transfermight be facilitated.

Possible evidence for a conformational rearrangement wasobtained by our previous observation of a small perturbationof the heme c spectrum when cytochrome c binds to theoxidase (11). The spectral change must be a reflection of achanging heme environment, since a direct heme-hemeinteraction can be ruled out (ref. 12 and references citedtherein). Here we report changes in CD and MCD spectra inthe Soret region when cytochrome c and oxidase form a tight1:1 complex.

EXPERIMENTAL PROCEDURESCytochrome c (horse) was Sigma type VI and was usedwithout further purification. Porphyrin-cytochrome c (13)and apocytochrome c (14) were prepared as described.Phosvitin (Sigma), poly(L-glutamate) (Fluka, average Mr =

32,500), and spermine (Fluka) were used as supplied. Beefheart cytochrome c oxidase was prepared according toHartzell and Beinert (15). The enzyme had 9 nmol of heme aper mg of protein and 18 mol of phosphorus per mol of hemeaa3. A d/A422 was 1.33 and A42x/AYID was 2.55. The maxi-mum turnover number with ferrocytochrome c was 170 s at20'C in 10 mM Tris.HCI/15 mM NaCl/0.05% dodecylmaltoside, pH 7.4. This buffer was used for all spectroscopicmeasurements. The enzyme was brought into this buffer byion-exchange chromatography (16). Concentrations of hemec and heme aa3 were determined as before (11).CD and MCD spectra were recorded at 25 ±+ 1C on a

JASCO (Tokyo) spectropolarimeter (model 500C). MCDspectra were recorded with a magnetic field of 1.5 tesla (T),parallel and antiparallel to the light beam. The scan rate was20 nm/min and the time constant 1 or 2 s; seven spectra wereaccumulated at both orientations of the magnetic field. The

Abbreviations: CD, circular dichroism; MCD, magnetic CD.*Present address: Institut fur Molekularbiologie und Biophysik,ETH-Honggerberg, CH-8093 Zurich, Switzerland.t"Heme c" indicates the heme of cytochrome c. "Heme a" and"heme a3" indicate the two spectroscopically different but chem-ically identical hemes of cytochrome c oxidase. "Heme aa3"indicates the sum of heme a and heme a3.

6687

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 84 (1987)

.-i20" D 20

F15 CD e10 E~~~~~~~~~~~10 10

0 0 ~

410 420 430 440 410 420 430 440

Wavelength (Om)

FIG. 1. CD spectra and difference CD spectra for fully oxidized(resting) oxidase and ferricytochrome c. (A) CD spectrum of theoxidase-ferricytochrome c complex ( ) and sum ofCD spectraof oxidase and cytochrome c ( . ). (B) Difference CD spectrumfor the oxidase-ferricytochrome c complex ( ), equivalent tothe difference between the spectra shown in A; difference CDspectrum of ferricytochrome c measured at pH 11 and pH 7.4( ). Control: CD spectrum of the oxidase and ferricytochromec in the presence of 300 mM NaCl minus the summed spectra of thecomponents (

signal-to-noise ratio was above 20 for CD and much higher forMCD measurements.

Difference CD and MCD spectra were recorded with thesample in a tandem cuvette of 4.375-mm light path in each ofthe two compartments. The two compartments were filledwith 1.00 ml each of equally concentrated solutions ofcytochrome c and oxidase, respectively. Spectra were mea-sured before and after mixing of the contents of the twocompartments, and difference spectra were obtained bysubtracting the second from the first spectrum. In controlexperiments it was shown that absorption, CD, and MCDspectra ofthe individual proteins remained unchanged duringthe time course of the experiment. Spectra of the fullyreduced hemoproteins were recorded in standard buffer thatcontained in addition S mM ascorbate and 60 ,uM N,N,N',N'-tetramethylphenylenediamine, under argon in a tightly sealedcuvette. Protein solutions were degassed and saturated withargon three times before being placed in the cuvette. Tofacilitate comparison with published spectra, CD is ex-pressed as molar elliptical polarization, [0] (degrees-cm2ldmol-1), and MCD as the difference absorption coefficientfor left versus right circularly polarized light normalized to anapplied magnetic field of 1 T, Ae/H (M cm T)-. DifferenceCD and MCD are expressed as A[0] and A(Ae/H), respec-tively. Molar ellipticities and difference absorption coeffi-cients are based on the concentrations of heme c and hemeaa3, respectively.

RESULTSThe CD spectrum of a 1:1 complex of fully oxidized (resting)oxidase and ferricytochrome c is significantly different from

Table 1. Spectroscopic titration of cytochrome c oxidase withferricytochrome c

A[0] at 414 nm, t(Ae/H) at 406 nm,Heme c/heme aa3* deg cm2 dmol-I (M cm.T)1

0.38 22,900 1.940.80 51,300 4.121.13 60,000 5.201.49 56,700 4.842.20 57,800 5.38

*Heme aa3 was fixed at 4.14 /.LM.

l 10

I

O

V-w _

25

I20 12115 11510 ;&j

0 E0

WavelnhI (nm)

FIG. 2. (A) Calculated CD spectrum obtained by subtracting theCD spectrum of free oxidase from the spectrum of the oxidase-fer-ricytochrome c complex ( ); CD spectrum offree ferricytochromec (-). (B) Calculated CD spectrum obtained by subtracting the CDspectrum of free ferricytochrome c from the spectrum of theoxidase-ferricytochrome c complex ( ); CD spectrum of freeoxidase (-).

the sum of the CD spectra of its components (Fig. LA). Thedifference CD spectrum (spectrum of the complex minussummed spectra of components, Fig. 1B) has a maximum at414 nm and a small minimum at 431 nm. The difference CDspectrum resembles that which was observed for "alkaline"minus "neutral" ferricytochrome c (Fig. 1B); the two con-formational isomers of ferricytochrome c are in reversibleequilibrium with a pKa around 9 (review in ref. 17). No CDchange was observed in the presence of 300 mM NaCI, inwhich cytochrome c does not bind to the oxidase (Fig. 1B).No difference CD spectrum could be observed in the regionofthe a and p bands ofthe hemes (not shown). The differencesignal saturates at a 1:1 ratio of cytochrome c to oxidase(Table 1).The specificity of the difference CD spectrum was tested

by comparing the CD spectra of 20 ,M cytochrome c in thepresence and absence of 2 AM phosvitin, 3 ,uM poly(L-glutamate), or 0.5 M NaCl. In no case was a differencedetected. Similarly, the CD spectrum of 6 ,uM oxidase wasnot changed by 6 ,M porphyrin-cytochrome c, 12 AMapocytochrome c, 0.5 M spermine, or 0.5 M NaCl.J Porphy-rin- and apocytochrome c as well as spermine are known tobind to the oxidase and to inhibit the reaction withcytochrome c, whereas phosvitin and polyglutamate competewith oxidase by binding to cytochrome c (18).The difference CD spectrum shown in Fig. 1B indicates a

conformational perturbation of both ferricytochrome c andoxidase hemes. The Soret absorption maxima of ferric hemec, a3, and a are at 408, 414, and 427 nm, respectively (19, 20).Since heme a is the first electron acceptor of ferrocyto-chrome c (21), heme a3 is not expected to contribute to thedifference CD spectrum. On the basis of this assumption, theindividual contributions of hemes c and a to the differenceCD spectrum were estimated in the following way. From theCD spectrum of the 1:1 complex (Fig. LA, solid line) thespectrum offree oxidase (Fig. 2B, dotted line) was subtractedto yield a calculated CD spectrum that is composed of thespectrum of bound cytochrome c plus the difference signaldue to bound oxidase (Fig. 2A, solid line). In the region below415 nm this calculated spectrum is essentially that of boundcytochrome c, since the contribution of the oxidase is small.A comparison of the calculated spectrum assigned to boundcytochrome c with that of free cytochrome c (Fig. 2A, solid

tA difference of about £40] = 5000 deg cm2 dmol-I would have beendetected under our experimental conditions.

6688 Biochemistry: Weber et A

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Proc. Natl. Acad. Sci. USA 84 (1987) 6689

- E'oX

EE ,

o x

_0 I

x

_ .

50

40

30

20

10

0

Wavelength (nm)

FIG. 3. CD spectrum of fully reduced oxidase-ferrocytochromec complex (-----). Sum of CD spectra of fully reduced oxidase andferrocytochrome c ( ). CD spectrum of the fully reducedoxidase-ferrocytochrome c complex minus the summed spectra ofthe components ( ). The scale on the right ordinate refers toellipticities above 440 nm.

and dotted lines below 420 nm) reveals that the minimum at417 nm of free cytochrome c is reduced or lost whencytochrome c binds to the oxidase. This follows from theobservation of a shoulder in the calculated spectrum as wellas from the observation of a less pronounced shoulder in thespectrum ofthe complex, as compared to the spectrum ofthesum of the components (Fig. 1A).§

Fig. 2B (solid line) shows a calculated CD spectrum thatwas obtained by subtracting the absolute spectrum of freecytochrome c (Fig. 2A, dotted line) from the spectrum of thecomplex (Fig. L4, solid line). Here, the region above 420 nmcorresponds, in essence, to the spectrum ofheme a of boundoxidase, since the difference signal due to bound cytochromec is small in this region. Fig. 2B indicates that, upon bindingof cytochrome c, the oxidase spectrum shifts to the blue andthe ellipticity decreases. This interpretation is confirmed bythe large trough in the calculated spectrum of Fig. 2A.Whereas the oxidase-ferricytochrome c complex may be

regarded as a virtual enzyme-product complex, ferrocyto-chrome c bound to fully reduced oxidase resembles anenzyme-substrate complex. The CD spectrum of the lattercomplex again differs significantly from the summed CDspectra ofthe components (Fig. 3). In the difference spectrumof Fig. 3 the contributions of ferrous hemes c and aa3 areeasily distinguished. Below 430 nm the spectrum can beattributed to ferrocytochrome c alone because the CD spec-trum of fully reduced oxidase is featureless in this region (8).The maximum ellipticity increase is around 415 nm, as in theoxidized complex. Thus, oxidized and reduced oxidase areinducing a similar increase of ellipticity in ferricytochrome c

and ferrocytochrome c, respectively, indicating similar con-formational rearrangements in both redox states. In contrast,ferrocytochrome c seems to induce little conformationalrearrangement in the fully reduced oxidase (right part of Fig.3), whereas ferricytochrome c affects the conformation ofoxidized oxidase (Figs. 1 and 2).

§Since the absorption maximum of heme a3 is at 414 nm (20), thedifference CD spectrum between 410 and 420 nm could result froman increase of heme a3 ellipticity. In this case the ellipticity of hemea3 would have to increase by about 80,000 deg-cm2.dmol'I in theoxidase-cytochrome c complex. This seems very unlikely if theheme a/CuA pair is the primary electron acceptor of ferrocyto-chrome c (21).

The conformational rearrangements may be confined to theprotein matrix, with the change of the induced Soret CDarising from variations in the symmetry of the heme envi-ronments. But the changes could as well translate to a directchange of the heme electronic structure, by affecting the ironand porphyrin orbitals. Direct evidence for a change in theheme c electronic structure was obtained by comparing theMCD spectrum of the 1:1 complex of ferricytochrome c andoxidase with the sum of the MCD spectra of the components(Fig. 4). A difference MCD spectrum was observed with amaximum at 408 nm and a minimum at 420 nm. The main,derivative-shaped peak ofthe difference MCD spectrum (Fig.4, solid line) is attributed to a change in the electronicstructure of heme c because the MCD signal of the oxidaseis small and does not cross the zero line below 420 nm(compare dashed and dotted lines of Fig. 4). The maximumof the difference MCD spectrum coincides with the point ofinflection of the S-shaped spectrum of free ferricytochromec, hence binding to the oxidase shifts the heme c MCD signalby about 1 nm to longer wavelengths. A contribution ofhemea to the difference MCD spectrum of Fig. 4 cannot be ruledout, but must be small. [Ferric high-spin heme a3 has noMCDsignal in the Soret region (22).] TheMCD difference spectrumis specific to the oxidase-cytochrome c complex; it could notbe elicited by phosvitin, polyglutamate, or a change of ionicstrength (not shown).A very similar picture emerges for the fully reduced

oxidase-ferrocytochrome c complex (Fig. 5). The mainfeature of the difference MCD spectrum is again due to achange of the heme c electronic structure (peak at 418 nm)and again indicates a slight red-shift in the spectrum ofboundferrocytochrome c. The main peak of fully reduced heme aa3is at 447 nm (Fig. 5, dotted line, and ref. 22). Any contributionof heme aa3 to the difference MCD spectrum of Fig. 5 musttherefore be small. Essentially the same Soret differenceMCD spectrum was also obtained with the complex com-posed of ferrocytochrome c and partially reduced cyanide-inhibited oxidase (not shown). Ferrocytochrome c depicts avery strong MCD band in the visible region of the spectrum(23), but we found no significant change in MCD between 500and 600 nm when ferrocytochrome c bound to the fullyreduced oxidase.

DISCUSSIONThe induced CD of a heme group is very sensitive tovariations in the symmetry of the heme environment (reviewin ref. 8). The change of rotational strength may have two

.50k 5I

F°04->

-50 .5

-100 _-10

410 420 430 440Wavelength (nm)

FIG. 4. MCD spectra of ferricytochrome c (--, left ordinate) andoxidized oxidase (-, left ordinate). MCD spectrum ofthe oxidase-fer-ricytochrome c complex minus the summed spectra of the components(-, right ordinate). Control: MCD spectrum of the oxidase andferricytochrome c in the presence of 300 mM NaCl minus the summedspectra of the components (-.-, right ordinate).

Biochemistry: Weber et al.

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Proc. Natl. Acad. Sci. USA 84 (1987)

7.5

5I!Z.

2.5e'5

Wavelength (nm)

FIG. 5. MCD spectra of ferrocytochrome c (---, left ordinate)and fully reduced oxidase (., left ordinate). Difference MCDspectrum for the complex composed of fully reduced oxidase andferrocytochrome c minus the summed spectra of the components(-, right ordinate).

causes. It may arise from different coupling between theheme prosthetic group and conformationally mobile proteinchromophores in free and complexed cytochrome c andoxidase. Second, the observed CD change may arise directlyfrom altered heme-protein interactions, leading to perturba-tion of the heme electronic structures. Such a perturbation ofthe heme itself is not a prerequisite for the observed changein CD. In the case of MCD, however, the dichroism, whichis induced by an externally applied magnetic field, is insen-sitive to protein conformational variations unless they direct-ly affect heme-protein interactions. Given this complemen-tarity of information deducible from the CD and MCDmeasurements, we may draw the following general conclu-sion: Binding of both ferri- and ferrocytochrome c to theoxidase induces a conformational rearrangement of theprotein that, in turn, alters the geometries of ferric andferrous heme c. The observed changes are highly specific tothe oxidase-cytochrome c complex. Saturation of the differ-ence CD and MCD signal at one heme c per heme aa3confirms the presence of a single cytochrome c electrontransfer site on mitochrondrial cytochrome c oxidase (11, 24).

Since the complexes studied are virtual electron transfercomplexes one might argue that the changes of conformationand heme geometry would not be observed with the transientcomplex of ferrocytochrome c and "working" (pulsed)oxidase during active turnover. We believe this to be a veryremote possibility in view of the large size of the spectro-scopic signals, which, moreover, originate at the "activesites," that is, the hemes and their environments.What is the actual nature of the conformational changes?

The question cannot be reasonably answered for cytochromec oxidase; there is too little structural information about thisenzyme. We therefore restrict the rest of the discussion tocytochrome c. The outstanding feature of the CD change forthe oxidase-ferricytochrome c complex is the decrease ofnegative ellipticity at 417 nm. Several different perturbationsof the cytochrome c conformation are known to induce asimilar change of the CD spectrum. The 417-nm CD band islost when ferricytochrome c rearranges to the "alkaline"conformation (8, 25) in which the methionine-80 sulfur boundto heme iron is replaced by a new ligand of uncertain nature(17, 26). However, lack of the 417-nm band need not indicatea loss of the Fe-S bond, since the band is also missing in thespectrum of the 1-65 heme peptide in the presence ofN-acetylmethionine (8). Partial denaturation of ferricyto-chrome c by different alcohols abolishes the 417-nm negativeellipticity (27) and turns the reduced protein autooxidizable(28). Finally, replacement of the invariant phenylalanine-87

of yeast iso-1-cytochrome c (equivalent to phenylalanine-82of horse cytochrome c) by serine, glycine, or tyrosine alsoeliminates the negative ellipticity at 417 nm (29).Common to these different perturbations of the native

cytochrome c structure is a conformational rearrangement ofthe heme environment. This leads us to postulate that bindingof cytochrome c to the oxidase induces a rearrangement ofthe heme environment, possibly in the area of the exposedheme edge, known to be part of the recognition site forcytochrome c (1), and on the methionine-80 side of the heme.A similar oxidase-induced rearrangement takes place inoxidized as well as in reduced cytochrome c.$

Perturbation of the heme environment by the oxidase ispredicted from an entirely different observation. In nativeferricytochrome c the stability of the closed pocket dependscritically on two bonds above and below the entrance to theheme cleft (32). The bonds are between lysine-13 and gluta-mate-90 and between lysine-79 and the main chain carboxylof residue 47. Breaking ofthese bonds parallels the pKa ofthe"alkaline" transition as well as the opening ofthe Fe-S axialbond during thermal denaturation (32). The same two bondsmay be opened in bound cytochrome c because lysine-13 andlysine-79 are both at the oxidase binding site (1-5, 33).

Is there a correlation between the conformational re-arrangement and the electron transfer function of cyto-chrome c? Alteration of the heme-protein interaction mayhelp to decrease the activation free energy of the electrontransfer reaction. Given a fixed overall free energy change(AG') and a fixed heme-heme distance, two parameterscrucially determine the activation free energy-namely, thenature of the protein matrix between the hemes and thereorganization free energy. The latter is necessary for thestructural rearrangement of heme and protein following theoxidation of ferrous heme c (6, 7, 34, 35). To facilitateelectron transfer, the geometry of heme c should be midwaybetween the geometries of the ferric and the ferrous statesand some rearrangement should take place before the elec-tron transfer (7, 34). Upon oxidation, the porphyrin under-goes flattening and is tilted (10). The angle between thenormal to the porphyrin plane and the Fe-S bond increasesby about 50 (10), weakening the Fe-S bond. One couldspeculate that the spectral changes reflect a similar yetsmaller movement. The conformation of the methionine-80side chain relative to the heme determines the distribution ofelectron spin density on the heme. It has been speculated thatthe interaction with a redox partner could trigger a rearrange-ment of the relative positions of heme and methionine-80axial ligand and thus of the distribution of electron spindensity, thereby facilitating electron transfer (36). Indeed,NMR studies on cytochrome c complexed to various electrondonors and acceptors do show such a redistribution of spindensity (37). The MCD change might arise, in part, from thisphenomenon.We have found only one previous report on a CD change

concomitant to complex formation of cytochrome c (38).Chiang et al. observed a CD difference between the cyto-chrome c-cytochrome c1 complex and the summed spectra of

MThe conformation of oxidase-bound cytochrome c is clearly differ-ent from the conformations of both native and "alkaline" cyto-chrome c. This follows from preliminary experiments withcytochrome c derivatives with a single nick in the polypeptidechain-e.g., between arginine-38 and lysine-39 (30). The pKa valuesfor the "alkaline" transition of these derivatives range from 7.1 to7.6 (31), hence they exist as mixtures of native and alkalineconformations at the pH of the spectroscopic binding experiment,7.4. The difference MCD signal observed with these derivatives isaccounted for by (i) a contribution due to an oxidase-imposedtransition from the "alkaline" to the native conformation and (it) acontribution due to the transition from the native to the oxidase-bound conformation (B.M., unpublished experiments).

6690 Biochemistry: Weber et al.

117,

II

'L.

P92

x

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Proc. Natl. Acad. Sci. USA 84 (1987) 6691

the two proteins. From their data (figure 3 of ref. 38) onecalculates a difference CD spectrum that is very similar to theone for the oxidase-cytochrome c complex shown in Fig. 1B.The spectroscopic studies should now be extended to otherelectron transfer complexes of cytochrome c, as well as toderivatives of cytochrome c, to further the understanding ofthe relationship between the recognition/binding process andthe electron transfer function.

We thank Drs. J. Kagi, M. Vasak, and W. H. Koppenol forstimulating discussions, Drs. A. Proudfoot and C. Wallace for a giftofsome cytochrome c derivatives, and Dr. B. Gutte for help with thepreparation of porphyrin-cytochrome c. This work was supported bySwiss National Science Foundation Grant 3.114.85, by the Hart-mann-Muller Stiftung, and by the Kanton of Zurich.

1. Margoliash, E. & Bosshard, H. R. (1983) Trends Biochem. Sci.8, 316-320.

2. Smith, H. T., Staudenmayer, N. & Millett, F. (1977) Biochem-istry 16, 4971-4974.

3. Ferguson-Miller, S., Brautigan, D. L. & Margoliash, E. (1978)J. Biol. Chem. 253, 149-159.

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