Eur. J. Biochem. 91, 255-261 (1978)

Circular-Dichroism Studies of the Cytochrome b-cl Complex of Saccharornyces cerevisiae Jennifer REED, Thomas A. REED, and Benno HESS

Max-Planck-Institut fur Ernahrungsphysiologie, Dortmund

(Received July 25, 1977/May 9, 1978)

1. Circular dichroism studies on the Soret region of the cytochrome b-cl complex of yeast reveal a change in the dichroism of cytochrome c1 depending on the redox state of cytochrome b, indicating a conformational interaction between both cytochromes.

2. This interaction is not influenced by binding of the inhibitor antimycin A to the complex, so that the interaction does not appear to be involved in the mechanism of electron transport through the complex.

3. Antimycin A binding causes a complex set of changes in the CD spectrum of the complex, which can be attributed to a severe and specific distortion of the environment of the chromophore of cytochrome b.

Recent work in a number of areas [l - 41 has under- lined the importance of conformational changes in protein tertiary structure to the control and the mech- anism of enzymic reactions. Several workers have suggested on theoretical grounds that such adjust- ments of protein structure might play a role in the complex interactions of electron carriers and linked ATP synthesis in the respiratory chain [5].

Of the methods available for studying conforma- tional change, one of the most direct is that of circular dichroism. CD studies have been carried out on a number of individual cytochromes [6.- 1-51. Theoretical interpretations and mathematical syntheses of such spectra [15,16] permit a relatively detailed understand- ing of the role of the protein and its amino acid side chains in the induction of dichroism in the heme. Studies on intact, functional respiratory chain com- plexes are rarer; the b-el complex in particular has not yet been examined by these techniques due to the difficulty of obtaining a high enough concentration of the complex proteins in soluble form.

An active, antimycin-A-sensitive cytochrome b-el complex has been isolated from yeast in this labora- tory [17]. The enzymatic purity, the activity and solu- bility of the preparation in aqueous media make it appropriate for circular dichroism studies in a varie- ty of oxidation states. In this report, evidence is pre- sented for conformational interaction between cyto- chromes b and c1 which appears linked to electron

Abbreviations. CD, circular dichroism

transport and its control during the function of com- plex 111 in the respiratory chain of mitochondria.

MATERIALS AND METHODS

Cytochrome b-cl complex was prepared from Suc- charomyces cerevisiae strain YF as previously de- scribed [17]. Soluble preparations containing 2- 3 mg protein/ml in 0.45 M sorbitol at pH 7.8 (at approxi- mately 8 nmol heme/mg protein) were used for analy- sis. Sodium ascorbate (crystalline) and sodium dithio- nite were obtained from Merck AG, Darmstadt, and antimycin A from Calbiochem.

Absorption spectra were taken in a Cary 14 spec- trophotometer. Low-temperature spectra were ob- tained in a Chance split-beam spectrophotometer. Circular dichroism spectra were measured on a Jobin Yvon dichrograph 111 in a quartz cuvette with a 0.5-cm light path at 25 "C. Subtraction of background, signal averaging and further processing were carried out by digitalizing the data through a Nicolet model 1074 instrument computer. The standard scanning conditions were at a rate of 0.05 nm/s, a sensitivity of A units/mm, a dwell time of 2 s and an external time constant of 0.05 Hz, low pass. Signal and display scale amplitude were variable. Spectra were taken of the ellipticities associated with absorbance in the Soret region and covered the range from 370 nm to 470 nm.

The relatively low solubility of the b-el complex preparation, (in the range of 2.0 - 3.0 mg protein/ml),

256 Circular Dichroism of Yeast h-ci

with a rather low chromophore concentration yields a low signal-to-noise ratio, which was met by suitable spectrum signal averaging technique and/or high time constant of instrumentation. High spectral ac- curacy was achieved by regular wavelength calibra- tion of the dichrograph 111 against xenon lines of the source, the dichroism peak of isoandrosterone and the dichroism peak of L-10-camphorsulfonic acid [I 81. The reliability of the spectral measurements and signal averaging technique, as well as the analogue printing and scaling device involving the interface between the Nicolet 1070 data computer and a Jobin Yvon 111, is illustrated by the consistency of circular dichroic spectra obtained during a period of over one year with 14 separate samples of the b-cl complex, repre- senting a total of eight individual preparations. The standard deviation of these measurements was 1 0.7 nm. The smallest difference between peak posi- tions in these studies, about 5 nm, is therefore well within the limits of resolution of the instrumentation system.

RESULTS

In order to establish a specific redox state of cyto- chromes h and c1 within the complex, use was made of the different reducing properties of dithionite and ascorbate on the system. Fig. 1 shows the difference spectrum (reduced minus oxidized) in the CI region of the dithionite-reduced b-cl complex [I 71 contrasted with the difference spectrum of the ascorbate-reduced complex. In the case of ascorbate reduction, the 555-nm peak is essentially unaltered from that ob- tained with dithionite in excess, indicating that the cytochrome c1 has been reduced. The 560-nm peak, however, is missing. Thus it can be concluded that cytochrome b has not undergone reduction but re- mains oxidized in the presence of ascorbate.

The circular dichroic spectrum of the oxidized cytochrome b-cl complex (Fig. 2) exhibits a positive ellipticity at 412.5 nm. This corresponds closely in ellipticity maximum, half-band width and molar ellip- ticity to the circular dichroism spectrum of the oxidized form of purified mammalian cytochrome c1 obtained by Yu et al. [6]. Although the oxidized form of cyto- chrome h has an absorbance maximum at 418 nm, there is no indication of a circular dichroic maximum in this region, or of any broadening of the 412.5-nm peak which might indicate a shoulder.

If the b-c1 complex is treated with excess sodium ascorbate, the C D spectrum shown in Fig.3 is obtained. The single peak at 418 nm coincides with the absorp- tion maximum of ferrocytochrome c1 and also agrees with the C D spectrum of isolated, reduced mammalian c1 [6]. This once again follows the simplest case extra- polation ; cytochrome b remains oxidized in the presence of ascorbate and makes little or no contri-

I ‘I

\/- I 01 I I 1 I 1 530 540 550 560 570 580

Wavelength (nrn)

Fig. 1 . Difference sprctru (reduced minus nxidixdJ of‘ fJw b-ct complex reduced with dithionite (-- - ~ - ) and uscorhutr i-~ )

412

I l l I l l , 1 1 4

380 400 420 440 460 Wavelength (nrn)

Fig. 2. CD sprctrum of oxidized cytochrome b-cl complex. 2.2 mg protein/ml. Cuvette vol. = 3.0 ml. Scanning speed = 0.05 nmjs. Sensitivity = 1 A unit/mm. Dwell time = 2.0 s. Display scale am- plitude = 128. External time constant = 0.05 Hz, low pass. Am- plitude = & 1 V. Signal average of four curves

bution to the C D spectrum as anticipated from its behaviour in Fig. 2 .

Dithionite reduction of the h-cl complex would thus be expected to result in a C D spectrum having

J. Reed, T. A. Reed, and B. Hess 257

3 i

I,,,,,,,,,, 380 400 420 440 460

Wavelength (nrn)

Fig. 3. CD spectrum of ascorbate-reduct,d cytochrome b-cl complex. Scanning conditions as in Fig. 1

one of two possible forms. On the one hand, a single ellipticity at 412nm (the absorption maximum of ferrocytochrome el) might be seen if ferrocytochrome h as well as ferricytochrome b made no contribution to the CD spectrum. Alternatively, a double system might be seen with peaks at 418 nm and also at or around 429 nm, the absorbance maximum of ferro- cytochrome h. What is actually observed (Fig. 4) resembles neither of these two simplest case possibili- ties. The CD spectrum exhibits a single positive ellip- ticity at 423 nm, a wavelength roughly between the absorbance maxima of the two cytochromes present. There is no indication of shoulders which might cor- respond to contributions from the 41 8-nm or 429-nm maxima. If dithionite is added subsequent to treatment with ascorbate, a spectrum identical to the dithionite- reduced spectrum is obtained.

In the presence of antimycin A, an inhibitor of electron transport between cytochromes h and el, the standard oxidized, ascorbate-reduced and dithio- nite-reduced CD spectra are all sharply modified. In all three spectra a large new positive peak is ob- served at about 419-420 nm. This is clearly seen in the case of the oxidized (Fig. 5 ) and dithionite-reduced (Fig. 6) spectra, where the satellite peak can be dif- ferentiated clearly from the 412.5-nm and 423-nm peaks normally present. In the case of the ascorbate- reduced spectrum (Fig. 7), no clear separation can be

380 400 420 440 460 Wavelength (nm)

Fig. 4. CD spectrum of dithionite-reduced cytochrome b-cl complex. Scanning conditions as in Fig. 1

380 400 420 440 460 Wavelength (nm)

Fig. 5. CD spectrum of oxidized cytochrome b-c, complex plus anti- mycin A . 1.6 nig protein/ml. 20 pl 20 mM antimycin A. Signal average of eight curves. Other scanning conditions as in Fig. 1

258

419 423

Circular Dichroism of Yeast b-cl

1

1 , , 1 1 1 , , 1 ,

380 400 420 440 460 Wavelength (nrn)

Fig. 6. CD spectrum of dithionite-reduced cytochrome b-cl complex plus antimycin 4 . Scanning conditions as in Fig. 5

seen between the new 419-420-nm peak and the 418-nm ellipticity due to ferrocytochronie CI. There are, however, certain differences in form between this curve and that of Fig. 3 which suggest that the sharper 419 - 420-nm peak may be superimposed on the exist- ing 418-nm one.

In order to exclude the possibility that these peaks arise as artefacts from antimycin A itself, high-sen- sitivity absorption spectra of antimycin A were ana- lyzed and showed no absorption maximum at 419- 420-nm; nor does a CD spectrum of the inhibitor in buffer indicate any ellipticity in this area which could be ascribed to the simple presence of antimycin A.

In addition, the dithionite-reduced spectrum ex- hibits a sharp negative ellipticity at 398 nm which differs in form and position from the broad negative ellipticity of cytochrome c1.

DISCUSSION

The circular dichroism of a chromophore may be intrinsic to the chromophore or induced by an asym- metric environment. The presence of absorption peaks in the cytochromes due to TC-TC* transitions of the porphyrin chromophore will not necessarily lead to a corresponding maximum in the CD spectrum since the

419

I I I I I I I I I L

380 400 420 440 460 Wavelength (nm)

Fig. I . CD spectrum of axorhate-reduced cytochrome b-cl complex plus actinomycitz A . Scanning conditions as in Fig. 5

porphyrin is itself symmetric. Circular dichroism in the visible and Soret regions is dependent on induction arising from an asymmetric protein environment [15]. Thus changes in the conformation of the affecting protein moieties will modify the CD spectrum of the cytochrome and may be monitored in this way [16].

The b-cl complex used in these studies is isolated with the cytochromes in the oxidized form [17]. Addition of dithionite affects total reduction of the cytochromes present. In the presence of ascorbate, however, only cytochrome c1 is reduced, while the b cytochrome(s) remain completely oxidized (Fig. 1) as also observed in plant mitochondria [19] and suc- cindte-cytochrome c reductase [20].

The position of Soret absorbance to be expected in the case of the components of the b-cl complex is therefore as follows: (a) for oxidized complex, a 410-nm peak due to ferricytochrome c1 and a 418-nm peak due to ferricytochrome b ; (b) for ascorbate- reduced complex, a 418-nm peak due to ferrocyto- chrome c1 and a 418-nm peak due to ferricytochrome 6; (c) for dithionite-reduced complex, a 418-nm peak due to ferrocytochrome c1 and a 429-nm peak due to ferrocytochrome b. The CD spectrum of the oxidized h-cl complex, as discussed in Results, shows only the ellipticity associated with cytochrome c1 absorbance, with no apparent contribution from ferricytochrome b even though it absorbs at 418 nm. The single-com-

J. Reed, T. A. Reed, and B. Hess 259

ponent nature of the 412.5-nm peak is confirmed by the near identity of its half-band width and molar ellipticity to that of isolated cytochrome c1. This means that within the limits of resolution, which the combined techniques of the Jobin Yvon optics and signal averaging have made very high, there is no observable dichroism associated with ferricytochrome 6.

On ascorbate reduction, the cytochrome b remains oxidized and so would continue to produce no ob- servable CD signal. The ellipticity maximum at 418 nm is associated with the absorbance maximum of ferro- cytochrome c1 and again corresponds closely in peak position, half-band width, and molar ellipticity to that of isolated cl . To summarize, where cytochrome b is in the oxidized form, (a) there is no cytochrome b contribution to the CD spectrum and (b) both ferri- cytochrome c~ and ferrocytochrome c~ behave in very much the same manner within the complex as they do in isolation.

On addition of sodium dithionite, cytochrome b as well as cytochrome c1 is fully reduced. With the change of cytochrome b from the oxidized to the re- duced form, the behavior of cytochrome c1 within the complex no longer resembles that in isolation. As noted in Results, rather than a single 418-nm peak, or peaks at 418 nm and 429 nm, one observes a single peak at 423 nm. That this is a single-component peak, rather than an overlap of two or more components, is a valid assumption for the following reasons.

(a) Analysis of the reproducibility and standard deviation of peak position shows that it is highly probable that the system can separately distinguish two peaks 5 nm apart at equal dichroic amplitude, still more the 1 1-nm separation between 418 nm and 429 nm.

(b) The half-band width of the 423-nm peak (17 nm) is comparable to that of single component CD peaks.

(c) Subtraction of the 41 8-nm ferrocytochrome el signal of the ascorbate-reduced complex from the 423-nm peak of the dithionite-reduced complex does not give rise to a 429-nm peak or anything directly associated with ferrocytochrome b absorbance. In- stead, one obtains a highly asymmetrical peak at 423 m i , exactly what one would expect if the 423-nm peak were a higher-amplitude single-component sig- nal. Subtraction of a synthetically produced 429-nm peak of similar amplitude to the 418-nm signal has similar results.

It is evident that the dichroic spectrum of ferro- cytochrome CI within the complex, once cytochrome b is also reduced, differs significantly from that obtained in isolation. The shift in ellipticity maximum from 418 nm to 423 nm points to an alteration in the confor- mation of ferrocytochrome c1 resulting from its inclu- sion in the complex, i.e. an interaction of c1 with one or more of the complex I11 proteins. On the other hand,

the CD spectrum of the b-cl complex in the oxidized and in the ascorbate-reduced states, in each of which cytochrome b remains oxidized, closely resembles that of isolated mammalian c1. Thus the conformatio- nal alteration indicated by the spectral shift seems to be dependent on the redox state of cytochrome b. Both for this reason and because local disymmetry effects extend only over short distances (1.5 nm [15]), it would seem that of the complex I11 proteins, cyto- chrome c1 is most probably interacting with cyto- chrome b.

It is unlikely that the effects causing this particular shift include a significant amount of heme-heme inter- action. Such coupling normally results in symmetrical distribution of positive and negative peaks across the absorbance maximum. While the angle between coupled chromophores can symmetrically alter the size of the resultant bilobe signal, and in the case of chromophore multilayers can induce asymmetry be- tween the respective lobes, it is improbable that com- plete suppression of one lobe and thus an apparent wavelength shift can be caused by angular effects in simple coupling models. It is more probable that what one is seeing here is the result of interactions between those portions of the polypeptide which induce dichro- ism in the heme. Given the dependence of the 418 nm to 423 nm shift on the redox state of cytochrome b, it would seem that reduction of ferricytochrome b results in an alteration in its tertiary structure which brings about interaction between the two proteins resulting in a conformational change in cytochrome c1. Alterations in the tertiary structure of the surround- ing peptide domain are known to be capable of in- ducing shifts in the CD spectrum of a heme chromo- phore, as in the case of cytochrome c [7] and cyto- chrome bz [8,21].

The indirect contribution of cytochrome b to the dithionite-reduced CD spectrum is of special interest. Difference spectra of the b-el complex used in these studies show unequivocally the presence of cytochrome b [17], yet there is no clearly resolved component in Fig. 4 which can be attributed to it. The interpretation of this phenomenon is dependent upon what type of CD spectrum can be expected of cytochrome b in isolation, a question which is unresolved at present. While microsomal cytochromes b from mammalian [9-111 and bacterial [ l l ] sources do possess dichro- ism in the Soret region, these are not as close, struc- turally, to mitochondria1 cytochromes b as cytochrome c is to cytochrome c l , and these last two differ appre- ciably in their dichroic properties. A very small con- tribution from cytochrome b566 has been detected in the CD spectra of whole electron transport particles from beef heart [22]. Unfortunately, this was not ob- servable in the spectra per se, but was only determined by the subtraction of spectra of the electron transfer particle in various redox states. No contribution of

260 Circular Dichroism of Yeast b-c1

cytochrome b561 to the CD spectrum of electron transfer particles was reported by these authors. Mitochondria1 cytochrome b, therefore, may be con- tributing only weakly to the dichroism of the b-el complex under normal conditions. While one is hesi- tant to state unequivocally that cytochrome b exhibits no dichroism, if it exists it is too weak to cause dis- cernable shoulders in the experiments of either Fig.2 or Fig.4. Its presence in the CD spectrum is only observed indirectly in its effects on cytochrome c1.

If the 418 nm to 423 nm shift is fundamental to the process of electron transfer between cytochromes b and c1, one would expect it to be affected by an inhibi- tor of electron transfer through site 11, such as anti- mycin A. Although the CD spectra of the b-cl com- plex given in Fig. 5 - 7 show a marked alteration in the presence of the inhibitor, the 418 nm to 423 nm shift does not appear to be affected. The dithionite-reduced spectrum (Fig. 6) maintains the characteristic 423-nm peak. It is difficult in the face of these data to justify any essential involvement of cytochrome c1 conforma- tional change in electron transport.

That binding of antimycin A does have an effect on some other aspect of protein conformation is indicated by the appearance of a new positive ellip- ticity at 419-420 nm in Fig. 5 and 6, and possibly in Fig.1. The redox state of the cytochromes has no influence on the antimycin-A-induced peak ; the effect is rather as though a peak at 419 nm had simply been added to the spectra of Fig.2-4. Antimycin A does not absorb at these wavelengths, nor has it any in- trinsic dichroism, therefore the 419-nm ellipticity is not due to simple addition of antimycin A. It arises as a direct result of binding to the complex and, as the previously described conformational change in cytochrome c1 is not affected, the site of antimycin attachment is probably not on the cytochrome c1 protein. From investigations on the reaction of anti- mycin A with reconstituted succinate-cytochrome c reductase, [23], it was concluded from the stoichio- metry of maximal inhibition that the inhibitor bound only to cytochrome b. The CD data reported indirectly support this conclusion.

The combination of a positive peak at 419 nm in all three curves plus a negative peak at 398 nm in the dithionite-reduced curve represents a complex set of interactions which does not lend itself to easy inter- pretation. The peak at 419 nm might at first glance be ascribed to the cytochrome c1 chromophore. The behavior of cytochrome c1 in the absence of antimycin A, however, has been described and does not differ appreciably when the inhibitor is present. Further- more, the 419-nm peak is present when all cytochromes are in the oxidized state and the maximum absorbance of the c1 heme is at 410 nm. On the other hand, neither the 419-nm nor the 398-nm peaks correspond to the maximum absorbance of ferrocytochrome b. If

one attempts to treat the region from 398-419 nm as an asymmetrical bilobal structure, then the zero intercept at 412 nm should correspond to the absorp- tion of the interacting chromophores, and none of the ferrocytochromes present has maximum absorbance at this wavelength. The additional extrema induced by antimycin A do not, in fact, correspond to the ab- sorption maxima of any chromophore present.

The presence of a dichroic peak in the apparent absence of a related absorption maximum is not en- tirely unknown, bovine rhodopsin [24] being an exam- ple. The cis peak in its CD spectrum has been inter- preted as the result of a highly strained and specific state of the chromophore. Such a condition could be involved in the mechanism by which antimycin A in- hibits electron transport between cytochrome b and CI ; indeed, antimycin A interference with a redox-medi- ated conformational change has been postulated [25].

A mechanism which might explain both the failure of the 419-nm peak to respond to changes in the redox state of the cytochromes and the possible strain in- duced on the chromophore is suggested by the follow- ing points: (a) the 419-nm dichroic peak associated with antimycin A binding corresponds to the absorp- tion maximum of ferricytochrome b (which is di- chroically ‘invisible’ under normal conditions) ; (b) the antimycin-A-induced maximum is noticably sharper than the standard CD peaks (the estimated half-band width is about 13 nm).

These phenomena of peak development and appa- rent increase in resolution resemble results obtained in dichroic studies of the aromatic regions of proteins at 77 K [26]. In the low-temperature studies, the in- creased resolution is believed to result from locking of the protein into a specific conformation which immobilizes the side chains and effectively removes the presence of slightly differing conformations which detract from the effect of the dominant form. An actual splitting of cytochrome b absorption bands at 77 K has been reported [27]. We suggest that antimycin A binding has locked one of the b cytochromes into a distorted but specific form, bringing about an effective amplification and sharpening of any ellipticity asso- ciated with it.

This leaves the question of the presumed ‘frozen b’ band remaining fixed at 419 nm despite the changes in oxidation state of the complex. This is reminiscent of an earlier analysis of the effect of antimycin A under anaerobic conditions in yeast suspensions, where an apparent oxidation of cytochrome h was ob- served [28]. The antimycin-A-locked conformation of the protein could be so strong as to be independent of the redox changes of the heme. Such an effect has recently been described for ferrocytochrome c and defined as a ‘pseudo-oxidized’ spectrum [29].

We are thus presenting a picture of antimycin A binding to one of the b cytochromes, probably not

J. Reed, T. A. Reed, and B. Hess 26 1

that involved in protein-protein interaction with cytochrome c1, and locking it into a highly specific, distorted form. This would account for the effects seen, is in accordance with phenomena observed in similar systems and provides an intriguing basis for a mechanism of antimycin A inhibition.

In summary, analysis of the circular dichroism spectra of the b-cl complex indicates that cytochrome CI is bound into the complex in such a way that an interaction occurs between the b and CI proteins which changes the conformation of cytochrome CI. This interaction is not affected by antimycin, an inhibitor of electron transport between cytochromes b and c1, and it is thus unlikely that it is central to the mechanism of electron transport through this site. The possibility that it is purely structural in nature, being a conse- quence of the close fitting of complex I11 proteins, is also unlikely in view of its dependence upon the redox state of cytochrome b. A functional change in conformation linked to electron transport through dependence on the redox state of cytochrome b imme- diately suggests a connection with the mechanism of energy conservation. The possibility of such a con- nection is currently under investigation in this labora- tory.

Binding of antimycin A to the complex results in the appearance of ‘independent’ CD peaks with high optical activity. This unusual phenomenon may be due to the induction of a highly strained form on the chromophore, or to a change in the electromagnetic environment of some specific portion of the protein. The effect of antimycin binding is to alter the con- formation of the b-cl complex.

We should like to thank Professor D. W. Wetlaufer for his helpful comments and criticism during the preparation of this paper.

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J. Reed, T. A. Reed, and B. Hess*, Max-Planck-Institut fur Erniihrungsphysiologie, Rheinlanddamnl 201, D-4600 Dortmund, Federal Republic of Germany

* To whom correspondence should be addressed