Proc. Natl. Acad. Sci. USA Vol. 76, No. 3, pp. 1045-1048, March 1979
Biochemistry
Circular dichroic evidence for a conformational change in a cytochrome b-cl complex by uncoupling agents (exciton splitting/heme-heme interactions/electron transfer/complex III/coupling mechanism)
JENNIFER REED, THOMAS A. REED, AND BENNO HESS Max-Planck-Institut fur Ernahrungsphysiologie, Rheinlanddamm 201, 4600 Dortmund, West Germany
Communicated by Henry Lardy, November 27, 1978
ABSTRACT Circular dichroic spectra of the cytochrome b-cl complex exhibit bilobe formation typical of exciton splitting in the presence of uncoupler. Bilobe formation occurs if both cytochrome cl and cytochrome b are fully reduced. The fully oxidized and ascorbate-reduced complexes are not altered dichroically by uncouplers. The exciton splitting induced by uncoupler is consistent with heme-heme interaction: specifically, interaction between the two cytochromes b in the complex. Recent work in this laboratory has revealed a conformational interaction between the cytochrome b and cytochrome cl proteins in a purified, active complex III preparation from yeast mitochondria (1). This interaction was dependent on the redox state of cytochrome b, taking place only when the b heme was in the reduced form. It was, however, unaffected by treatment with antimycin A, which inhibits up to 90% of electron transport in this preparation (2), and so is unlikely to be central to the normal mechanism of electron transport per se. A functional change in conformation linked to electron transport through dependence on a redox state of cytochrome b suggests some connection with the mechanisms of energy conservation. The possible involvement of conformational change of electron transport carriers in the events linking electron transport and ATP synthesis has received attention recently from several groups. Accordingly, the effect on the cytochrome b/cytochrome cl interaction of various compounds that act as uncouplers of oxidative phosphorylation was examined. The results of these investigations are reported here.
MATERIALS AND METHODS Cytochrome b-cl complex (complex III) was prepared from Saccharomyces cerevisae YF as described (2). Circular dichroism (CD) spectra were obtained by means of a Jobin Yvon Dichrograph III equipped with a repetitive device. Spectra were taken of the ellipticities associated with Soret region absorbance peaks of the cytochromes and covered the region from 470 to 370 nm. Bilobe spectra were the average of 16 scans at 25°C in a quartz cuvette with a 0.5-cm light path and a 3-ml volume. Signal averaging and background removal were carried out digitally with a Nicolet Instrument Corporation (Madison, WI), model 1074 instrument computer (1). All bilobe spectra were measured at least twice on separate days with fresh preparations of complex III. The average of all midpoint values with uncouplers was 430.8 + 0.5 nm. Continuous instrument standardization was conducted with isoandrosterone or the xenon lamp emission lines or both. The CD peak of the oxidized particle had a standard deviation smaller than 0.5 nm, based on 14 measurements from eight different 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.
mitochondrial preparations during a period of more than 1 year
(1).
Uncouplers were added to the cuvette as a 20-,ul aliquot, reaching a final concentration of 6.66 ,uM. This is in an approximate 1.5:1 stoichiometry with the b heme and appears to be saturating, as doubling the concentration produced no further effect. A small asymmetric scattering artifact between about 400 and 370 nm appeared on introduction of uncoupler into the cuvette. This was due to uncoupler forming a fine suspension when the alcoholic solvent was diluted with water. This scattering artifact was visible only because of the large number of "repeats" and high sensitivity of the measurements. The extent of this artifact was calculated as the difference between "oxidized" and "oxidized plus uncoupler" curves for each sample and subtracted from the "reduced plus uncoupler" curves to give the underlying spectrum. Curves treated in this manner were used to determine the crossover point of the bilobe (see Fig. 7). 2,4-Dinitrophenol and sodium arsenate were obtained from Fa. E. Merck AG (Darmstadt, Germany). Carbonylcyanide p-trifluoromethoxyphenol hydrazone was purchased from Sigma, dicoumarol from Boehringer Mannheim, and rotenone from EGA-Chemie KG (Ulm, Germany). RESULTS Fig. 1 shows the CD spectrum of the cytochrome b-cI complex in the fully oxidized state with and without added uncoupler. As can be seen from the almost complete superposition of the curves, the addition of uncoupler had no effect on the CD spectrum of the ferricytochromes in the complex. The behavior of all four uncouplers used was virtually identical, and only dicoumarol is shown here with the oxidized complex. Fig. 2 shows the normal CD spectrum of the fully reduced b-cl complex. The prominent positive ellipticity at 423 nm has been previously described (1) as due to a conformational change in the cytochrome cl protein brought about by interaction with reduced, and only with reduced, cytochrome b. When the b-cl complex was reduced in the presence of uncoupler (Figs. 3-6), the CD spectrum was altered. The single positive peak of Fig. 2 was replaced by positive and negative ellipticity maxima. The precise position of the positive and negative extrema varied slightly depending on the uncoupler used (see Table 1), but the crossover point in all cases lay at 430 nm. Inversion and superposition of the peaks shows that in all cases they were very nearly symmetrical (Fig. 7) although the amplitude of the negative ellipticity was somewhat greater than that of the positive. This inequality is due to overlap of the positive extremum with a masked, low-amplitude negative extremum at 402 nm (Fig. 7). Allowing for the distortion due to asymmetrical Abbreviation: CD, circular dichroism.
1045
1046
Biochemistry:
co
~
Reed et al.
Proc. Nati. Acad. Sci. USA 76 (1979)
I
0-1 _
370C
410
390
430
470
450
370
Wavelength, nm
FIG. 1. CD spectra of the oxidized (-) and oxidized plus dicoumarol (- .) b-cI complex; 2.0 mg of protein per ml. Path length = 0.5 cm. Scanning speed = 0.5 nm/sec. Sensitivity = 1 X 10-6 OD/mm. Time constant = 2 sec. Dwell time = 2.0 sec. Display scale amplitude = 1024. Amplification = +1 V. Signal average of 16 curves.
scattering at lower wavelengths, the system resembles a classical bilobe structure. Where ascorbate was used as reductant, only cytochrome cl was reduced; the b cytochrome(s) remained oxidized (1). The CD spectrum of cytochrome b-c1 complex reduced with ascorbate in the presence of uncoupler did not differ from the simple ascorbate-reduced spectrum reported previously (1). The distinct bilobe structure did not develop. The appearance. of the bilobe thus seems to be dependent on the heme of cytochrome b being in the reduced state. The bilobe structure is initiated only in the presence of uncoupler. Antimycin A, which binds to the b-cl complex and has a specific inhibitory effect, also has a typical effect on the CD spectrum which does not include bilobe formation (1). Rotenone, an inhibitor of electron transport at site I, has a multiple ring structure superficially similar to uncouplers such as dicoumarol. It has no effect on the CD spectrum of the complex. DISCUSSION The presence of a bilobe in CD spectra is due to the phenomenon of exciton splitting, in this case most probably as a result of heme-heme interaction. Exciton splitting occurs when two groups with overlapping absorption bands become juxtaposed Table 1. Variation of position of positive and negative extrema as a function of uncoupler
Uncoupler Dinitrophenol Arsenate Dicoumarol FCCP* *
Positive@, nm
Negative 0,
Crossover,
426.5 426.25 425 425
433.5 433.4 435.35 434.4
431 430.5 431 430.25
nm
Carbonylcyanide p-trifluoromethoxypenol hydrazide.
nm
410
390
430
450
470
Wavelength, nm
FIG. 2. CD spectrum of dithionite-reduced cytochrome b-c1 complex; 2.2 mg of protein per ml. Path length = 0.5 cm. Scanning speed = 0.05 nm/sec. Sensitivity = 1 X 10-6 OD/mm. Dwell time = 2.0 sec. Display scale amplitude = 128. External time constant = 0.5 Hz, low pass. Amplitude = t 1 V. Signal average of four curves.
(3, 4). This results in coupling between transitions at the same energy level, producing a reciprocal relation, one group having a positive and the other a negative rotational strength. As the ideal case, the positive and the negative extrema will be of equal magnitudes, with the isoelliptical point, where coupling is between identical chromophores, falling at the absorption maxi-
10A
-50
C -105 -20
-25 370
390
410 430 Wavelength, nm
450
470
FIG. 3. CD spectrum of dithionite-reduced complex plus 2,4dinitrophenol; 3.1 mg of protein per ml. Scanning conditions as in Fig. 1 except display scale amplitude = 4096.
Biochemistry:
Reed et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
1047
11 L
-1
2
CD
-'-3 4
390
410 430 Wavelength, nm
450
470
FIG. 4. CD spectrum of dithionite-reduced complex plus carbonylcyanide p-trifluoromethoxyphenol hydrazide; 2.9 mg of protein per ml. Scanning conditions as in Fig. 1 except display scale amplitude =
2048.
mum. Such a complex CD band, however, is usually superimposed on a spectrum of other transitions, so that perfectly symmetrical extrema are seldom observed. The bilobe observed closely resembles the general class of bilobes developing out of heme-heme interaction in model systems (5, 6). The spectrum in our case, however, has only two extrema as in the heme octapeptide derived from cytochrome c (6), as opposed to three extrema as shown by the corresponding heme undecapeptide (5). This strongly suggests that the b hemes in complex III are oriented parallel to each other in the manner of the front and back covers of a book.
Wavelength, nm
FIc. 6. CD spectrum of dithionite-reduced complex plus arsenate; 2.5 mg of protein per ml. Scanning conditions as in Fig. 1.
The transitions in the region 370-470 nm are due to the heme chromophores. While, theoretically, interaction between a Soret absorption band transition and one from, say, aromatic amino side chains is possible, the strength of exciton splitting falls off sharply with spectral distance between the bands. For this reason, the bilobe observed can be ascribed with a fair degree of confidence to heme-heme interaction, as the magnitude of
5 4
3 2 0 -1
C,!-2 -3
_
CD3 -4
-5
-5
-6 -7 -8 9
-
370
390
4 50 410 430 470 Wavelength, nm FIG. 5. CD spectrum of dithionite-reduced complex plus dicoumarol; 2.0 mg of protein per ml. Scanning conditions as in Fig. 1.
Wavelength, nm FIG. 7. CD spectrum of dithionite-reduced complex plus dicoumarol corrected for scattering. Dashed curve indicates symmetrical inversion of negative peak.
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Biochemistry:
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the extrema suggests a high degree of overlap in the absorption bands involved. Given the generally agreed content of one cytochrome cl heme and two b hemes per complex, possible interactions are restricted to those between either one b and one cl heme or between two b hemes. The close correspondence of the inflection point at 430 nm to the 429 nm absorption maximum of ferrocytochrome b would indicate a b-b interaction. The attribution of the heme-heme interaction to b cytochromes is partially dependent on the accuracy of extrapolation of the isoelliptical point after correction for scattering effects. The method used produced curves of similar form in all the "reduced plus uncoupler" spectra treated, eliminated the strong negative increase toward lower wavelengths, and revealed a broad negative extremum at about 402 nm. This similarity of form, and the occurrence of the newly resolved extremum at the same wavelength in treated curves from all four uncouplers, would seem to show that the scattering correction treatment gives an accurate representation of the underlying curve. Preliminary visualization of the molecular architecture of the cytochrome b-cl complex has been achieved by successive stepwise dissociation (7). Within each b-c1 unit, the two b molecules are adjacent to one another and span the phospholipid bilayer of the membrane with the contact axis of the cytochromes b normal to the plane of the membrane. This being so, any arrangement of the remaining proteins of the complex must be such as to increase the effective diameter of the complex in the plane of the membrane. In order to achieve exciton interaction, two hemes must approach within 10-15 A of one another (5), 15 A being the extreme outer limit of the possible range. This is most likely to occur between hemes of adjacent cytochromes b within the same complex, since the hemes b from different complexes are separated by at least twice the radius of a particle containing 250,000 daltons of protein plus 30% associated lipid. Such a distance is roughly an order of magnitude too great for the interaction observed to take place. The induction by uncoupling agents of heme-heme interaction between two b cytochromes could occur in two ways. The work of Erecinska-et al. (8) has shown that the respiratory chain hemes of mitochondrial membranes tend to be aligned perpendicular to the plane of the membrane. Within this configuration, however, the planes of two hemes might be in an orientation anywhere from mutually parallel, giving the highest probability of transition coupling, to mutually perpendicular, with very low probability. There is no clear evidence as to which of these configurations is most closely approximated by the b hemes. If in the normally coupled membrane they are mutually parallel but at too great a distance for interaction, binding of uncoupler may act by effectively reducing the distance between heme centers. If, on the other hand, two hemes are closely adjacent but mutually perpendicular, uncoupler binding may result in a change of angle, allowing transitional coupling to occur. Such changes in distance or angle would have to be mediated through conformational change in the surrounding protein. This in turn can be coupled to changes in the oxidation state of a heme (9). The occurrence of heme-heme interactions (4,9, 10), or what is otherwise interpreted as a major change in relative a-a3 heme
Proc. Natl. Acad. Sci. USA 76 (1979)
orientation (11, 12) in the case of cytochrome c oxidase, suggests that such a change may be involved in mediating electron transfer, especially as these effects are typical of only a specific set of redox conditions. It thus seems significant that circular dichroic indications of heme-heme interactions between b cytochromes do not occur in the b-cI complex under normally controlled conditions, but only in the presence of uncoupler. The induction of a specific uncoupler effect in the pure b-c1 complex is an unexpected result for several reasons. First, the b-cl complex as prepared is not a closed membrane system. Second, it does not include an active ATPase. Finally, sodium arsenate had an effect similar to that of the other compounds used, although it is not a "classical" uncoupler and appears to act through competitive inhibition of ATPase. Thus, the effect observed is, at least, something outside of or in addition to mechanisms acting through breakdown of a pH gradient. While one would hesitate to use these data to justify a separate "transitional coupling" theory of uncoupler action, it is clear both from the specificity of the bilobe formation and from the similarity of the curves produced by four chemically dissimilar uncoupling compounds, that there is more going on during the process than expected from a straightforward induction of change in membrane proton permeability. Relevant to this are the ideas presented in recent reviews on the possibility of conformational coupling of ATP synthesis and electron transfer in the mitochondrial membrane (13, 14). As pointed out in those articles, because the energy involved in conformational changes is sufficient to drive phosphorylation and because these changes are "likely to be accompanied by .... movement of protonated groups," both the normal function and the disruption of energy coupling may be mechanistically complex, involving both control of proton movement and specific conformational alteration in respiratory chain proteins. The results presented here would seem to support the view that the mechanism of uncoupling, at least, involves conformational phenomena as well as those of proton translocation. 1. Reed, J., Reed, T. A. & Hess, B. (1978) Eur. J. Biochem. 91, 255-261. 2. Reed, J. & Hess, B. (1977) Hoppe-Seyler's Z. Physiol. Chem. 385, 1119-1124. 3. Urry, D. W. (1965) Proc. Natl. Acad. Sci. USA 54,640-648. 4. Urry, D. W. & van Gelder, B. F. (1968) in Structure and Functions of Cytochromes, eds. Okenuki, K., Kamen, M. B. & Sekuzo, I. (Univ. of Tokyo Press, Tokyo), pp. 210-214. 5. Urry, D. W. (1967) J. Am. Chem. Soc. 89,4190-4196. 6. Urry, D. W. & Pettegrew, J. W. (1967) J. Am. Chem. Soc. 89, 5276-5283. 7. Weiss, H. (1978) in Energy Conservation in Biological Membranes, 29th Mosbacher Colloquium (Springer, Heidelberg, West Germany), pp. 31-42. 8. Erecinska, M., Blasie, J. K. & Wilson, D. F. (1977) FEBS Lett.
76,235-240.
9. Perutz, M. F. (1970) Nature (London) 228,726-739. 10. Tiesjema, R. H. & van Gelder, B. F. (1974) Biochim. Biophys. Acta 347, 202-214. 11. Palmer, G., Babcock, G. T. & Vickery, L. E. (1976) Proc. Nati. Acad. Sci. USA 73,2206-2210. 12. Babcock, G. T., Vickery, L. E. & Palmer, G. (1976)J. Biol. Chem.
251,7907-7919. 13. Boyer, P. D. (1977) Annu. Rev. Biochem. 46,955-1026. 14. Slater, E. C. (1977) Annu. Rev. Biochem. 46, 1014-1026.
Biochemistry
Circular dichroic evidence for a conformational change in a cytochrome b-cl complex by uncoupling agents (exciton splitting/heme-heme interactions/electron transfer/complex III/coupling mechanism)
JENNIFER REED, THOMAS A. REED, AND BENNO HESS Max-Planck-Institut fur Ernahrungsphysiologie, Rheinlanddamm 201, 4600 Dortmund, West Germany
Communicated by Henry Lardy, November 27, 1978
ABSTRACT Circular dichroic spectra of the cytochrome b-cl complex exhibit bilobe formation typical of exciton splitting in the presence of uncoupler. Bilobe formation occurs if both cytochrome cl and cytochrome b are fully reduced. The fully oxidized and ascorbate-reduced complexes are not altered dichroically by uncouplers. The exciton splitting induced by uncoupler is consistent with heme-heme interaction: specifically, interaction between the two cytochromes b in the complex. Recent work in this laboratory has revealed a conformational interaction between the cytochrome b and cytochrome cl proteins in a purified, active complex III preparation from yeast mitochondria (1). This interaction was dependent on the redox state of cytochrome b, taking place only when the b heme was in the reduced form. It was, however, unaffected by treatment with antimycin A, which inhibits up to 90% of electron transport in this preparation (2), and so is unlikely to be central to the normal mechanism of electron transport per se. A functional change in conformation linked to electron transport through dependence on a redox state of cytochrome b suggests some connection with the mechanisms of energy conservation. The possible involvement of conformational change of electron transport carriers in the events linking electron transport and ATP synthesis has received attention recently from several groups. Accordingly, the effect on the cytochrome b/cytochrome cl interaction of various compounds that act as uncouplers of oxidative phosphorylation was examined. The results of these investigations are reported here.
MATERIALS AND METHODS Cytochrome b-cl complex (complex III) was prepared from Saccharomyces cerevisae YF as described (2). Circular dichroism (CD) spectra were obtained by means of a Jobin Yvon Dichrograph III equipped with a repetitive device. Spectra were taken of the ellipticities associated with Soret region absorbance peaks of the cytochromes and covered the region from 470 to 370 nm. Bilobe spectra were the average of 16 scans at 25°C in a quartz cuvette with a 0.5-cm light path and a 3-ml volume. Signal averaging and background removal were carried out digitally with a Nicolet Instrument Corporation (Madison, WI), model 1074 instrument computer (1). All bilobe spectra were measured at least twice on separate days with fresh preparations of complex III. The average of all midpoint values with uncouplers was 430.8 + 0.5 nm. Continuous instrument standardization was conducted with isoandrosterone or the xenon lamp emission lines or both. The CD peak of the oxidized particle had a standard deviation smaller than 0.5 nm, based on 14 measurements from eight different 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.
mitochondrial preparations during a period of more than 1 year
(1).
Uncouplers were added to the cuvette as a 20-,ul aliquot, reaching a final concentration of 6.66 ,uM. This is in an approximate 1.5:1 stoichiometry with the b heme and appears to be saturating, as doubling the concentration produced no further effect. A small asymmetric scattering artifact between about 400 and 370 nm appeared on introduction of uncoupler into the cuvette. This was due to uncoupler forming a fine suspension when the alcoholic solvent was diluted with water. This scattering artifact was visible only because of the large number of "repeats" and high sensitivity of the measurements. The extent of this artifact was calculated as the difference between "oxidized" and "oxidized plus uncoupler" curves for each sample and subtracted from the "reduced plus uncoupler" curves to give the underlying spectrum. Curves treated in this manner were used to determine the crossover point of the bilobe (see Fig. 7). 2,4-Dinitrophenol and sodium arsenate were obtained from Fa. E. Merck AG (Darmstadt, Germany). Carbonylcyanide p-trifluoromethoxyphenol hydrazone was purchased from Sigma, dicoumarol from Boehringer Mannheim, and rotenone from EGA-Chemie KG (Ulm, Germany). RESULTS Fig. 1 shows the CD spectrum of the cytochrome b-cI complex in the fully oxidized state with and without added uncoupler. As can be seen from the almost complete superposition of the curves, the addition of uncoupler had no effect on the CD spectrum of the ferricytochromes in the complex. The behavior of all four uncouplers used was virtually identical, and only dicoumarol is shown here with the oxidized complex. Fig. 2 shows the normal CD spectrum of the fully reduced b-cl complex. The prominent positive ellipticity at 423 nm has been previously described (1) as due to a conformational change in the cytochrome cl protein brought about by interaction with reduced, and only with reduced, cytochrome b. When the b-cl complex was reduced in the presence of uncoupler (Figs. 3-6), the CD spectrum was altered. The single positive peak of Fig. 2 was replaced by positive and negative ellipticity maxima. The precise position of the positive and negative extrema varied slightly depending on the uncoupler used (see Table 1), but the crossover point in all cases lay at 430 nm. Inversion and superposition of the peaks shows that in all cases they were very nearly symmetrical (Fig. 7) although the amplitude of the negative ellipticity was somewhat greater than that of the positive. This inequality is due to overlap of the positive extremum with a masked, low-amplitude negative extremum at 402 nm (Fig. 7). Allowing for the distortion due to asymmetrical Abbreviation: CD, circular dichroism.
1045
1046
Biochemistry:
co
~
Reed et al.
Proc. Nati. Acad. Sci. USA 76 (1979)
I
0-1 _
370C
410
390
430
470
450
370
Wavelength, nm
FIG. 1. CD spectra of the oxidized (-) and oxidized plus dicoumarol (- .) b-cI complex; 2.0 mg of protein per ml. Path length = 0.5 cm. Scanning speed = 0.5 nm/sec. Sensitivity = 1 X 10-6 OD/mm. Time constant = 2 sec. Dwell time = 2.0 sec. Display scale amplitude = 1024. Amplification = +1 V. Signal average of 16 curves.
scattering at lower wavelengths, the system resembles a classical bilobe structure. Where ascorbate was used as reductant, only cytochrome cl was reduced; the b cytochrome(s) remained oxidized (1). The CD spectrum of cytochrome b-c1 complex reduced with ascorbate in the presence of uncoupler did not differ from the simple ascorbate-reduced spectrum reported previously (1). The distinct bilobe structure did not develop. The appearance. of the bilobe thus seems to be dependent on the heme of cytochrome b being in the reduced state. The bilobe structure is initiated only in the presence of uncoupler. Antimycin A, which binds to the b-cl complex and has a specific inhibitory effect, also has a typical effect on the CD spectrum which does not include bilobe formation (1). Rotenone, an inhibitor of electron transport at site I, has a multiple ring structure superficially similar to uncouplers such as dicoumarol. It has no effect on the CD spectrum of the complex. DISCUSSION The presence of a bilobe in CD spectra is due to the phenomenon of exciton splitting, in this case most probably as a result of heme-heme interaction. Exciton splitting occurs when two groups with overlapping absorption bands become juxtaposed Table 1. Variation of position of positive and negative extrema as a function of uncoupler
Uncoupler Dinitrophenol Arsenate Dicoumarol FCCP* *
Positive@, nm
Negative 0,
Crossover,
426.5 426.25 425 425
433.5 433.4 435.35 434.4
431 430.5 431 430.25
nm
Carbonylcyanide p-trifluoromethoxypenol hydrazide.
nm
410
390
430
450
470
Wavelength, nm
FIG. 2. CD spectrum of dithionite-reduced cytochrome b-c1 complex; 2.2 mg of protein per ml. Path length = 0.5 cm. Scanning speed = 0.05 nm/sec. Sensitivity = 1 X 10-6 OD/mm. Dwell time = 2.0 sec. Display scale amplitude = 128. External time constant = 0.5 Hz, low pass. Amplitude = t 1 V. Signal average of four curves.
(3, 4). This results in coupling between transitions at the same energy level, producing a reciprocal relation, one group having a positive and the other a negative rotational strength. As the ideal case, the positive and the negative extrema will be of equal magnitudes, with the isoelliptical point, where coupling is between identical chromophores, falling at the absorption maxi-
10A
-50
C -105 -20
-25 370
390
410 430 Wavelength, nm
450
470
FIG. 3. CD spectrum of dithionite-reduced complex plus 2,4dinitrophenol; 3.1 mg of protein per ml. Scanning conditions as in Fig. 1 except display scale amplitude = 4096.
Biochemistry:
Reed et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
1047
11 L
-1
2
CD
-'-3 4
390
410 430 Wavelength, nm
450
470
FIG. 4. CD spectrum of dithionite-reduced complex plus carbonylcyanide p-trifluoromethoxyphenol hydrazide; 2.9 mg of protein per ml. Scanning conditions as in Fig. 1 except display scale amplitude =
2048.
mum. Such a complex CD band, however, is usually superimposed on a spectrum of other transitions, so that perfectly symmetrical extrema are seldom observed. The bilobe observed closely resembles the general class of bilobes developing out of heme-heme interaction in model systems (5, 6). The spectrum in our case, however, has only two extrema as in the heme octapeptide derived from cytochrome c (6), as opposed to three extrema as shown by the corresponding heme undecapeptide (5). This strongly suggests that the b hemes in complex III are oriented parallel to each other in the manner of the front and back covers of a book.
Wavelength, nm
FIc. 6. CD spectrum of dithionite-reduced complex plus arsenate; 2.5 mg of protein per ml. Scanning conditions as in Fig. 1.
The transitions in the region 370-470 nm are due to the heme chromophores. While, theoretically, interaction between a Soret absorption band transition and one from, say, aromatic amino side chains is possible, the strength of exciton splitting falls off sharply with spectral distance between the bands. For this reason, the bilobe observed can be ascribed with a fair degree of confidence to heme-heme interaction, as the magnitude of
5 4
3 2 0 -1
C,!-2 -3
_
CD3 -4
-5
-5
-6 -7 -8 9
-
370
390
4 50 410 430 470 Wavelength, nm FIG. 5. CD spectrum of dithionite-reduced complex plus dicoumarol; 2.0 mg of protein per ml. Scanning conditions as in Fig. 1.
Wavelength, nm FIG. 7. CD spectrum of dithionite-reduced complex plus dicoumarol corrected for scattering. Dashed curve indicates symmetrical inversion of negative peak.
1048
Biochemistry:
Reed et al.
the extrema suggests a high degree of overlap in the absorption bands involved. Given the generally agreed content of one cytochrome cl heme and two b hemes per complex, possible interactions are restricted to those between either one b and one cl heme or between two b hemes. The close correspondence of the inflection point at 430 nm to the 429 nm absorption maximum of ferrocytochrome b would indicate a b-b interaction. The attribution of the heme-heme interaction to b cytochromes is partially dependent on the accuracy of extrapolation of the isoelliptical point after correction for scattering effects. The method used produced curves of similar form in all the "reduced plus uncoupler" spectra treated, eliminated the strong negative increase toward lower wavelengths, and revealed a broad negative extremum at about 402 nm. This similarity of form, and the occurrence of the newly resolved extremum at the same wavelength in treated curves from all four uncouplers, would seem to show that the scattering correction treatment gives an accurate representation of the underlying curve. Preliminary visualization of the molecular architecture of the cytochrome b-cl complex has been achieved by successive stepwise dissociation (7). Within each b-c1 unit, the two b molecules are adjacent to one another and span the phospholipid bilayer of the membrane with the contact axis of the cytochromes b normal to the plane of the membrane. This being so, any arrangement of the remaining proteins of the complex must be such as to increase the effective diameter of the complex in the plane of the membrane. In order to achieve exciton interaction, two hemes must approach within 10-15 A of one another (5), 15 A being the extreme outer limit of the possible range. This is most likely to occur between hemes of adjacent cytochromes b within the same complex, since the hemes b from different complexes are separated by at least twice the radius of a particle containing 250,000 daltons of protein plus 30% associated lipid. Such a distance is roughly an order of magnitude too great for the interaction observed to take place. The induction by uncoupling agents of heme-heme interaction between two b cytochromes could occur in two ways. The work of Erecinska-et al. (8) has shown that the respiratory chain hemes of mitochondrial membranes tend to be aligned perpendicular to the plane of the membrane. Within this configuration, however, the planes of two hemes might be in an orientation anywhere from mutually parallel, giving the highest probability of transition coupling, to mutually perpendicular, with very low probability. There is no clear evidence as to which of these configurations is most closely approximated by the b hemes. If in the normally coupled membrane they are mutually parallel but at too great a distance for interaction, binding of uncoupler may act by effectively reducing the distance between heme centers. If, on the other hand, two hemes are closely adjacent but mutually perpendicular, uncoupler binding may result in a change of angle, allowing transitional coupling to occur. Such changes in distance or angle would have to be mediated through conformational change in the surrounding protein. This in turn can be coupled to changes in the oxidation state of a heme (9). The occurrence of heme-heme interactions (4,9, 10), or what is otherwise interpreted as a major change in relative a-a3 heme
Proc. Natl. Acad. Sci. USA 76 (1979)
orientation (11, 12) in the case of cytochrome c oxidase, suggests that such a change may be involved in mediating electron transfer, especially as these effects are typical of only a specific set of redox conditions. It thus seems significant that circular dichroic indications of heme-heme interactions between b cytochromes do not occur in the b-cI complex under normally controlled conditions, but only in the presence of uncoupler. The induction of a specific uncoupler effect in the pure b-c1 complex is an unexpected result for several reasons. First, the b-cl complex as prepared is not a closed membrane system. Second, it does not include an active ATPase. Finally, sodium arsenate had an effect similar to that of the other compounds used, although it is not a "classical" uncoupler and appears to act through competitive inhibition of ATPase. Thus, the effect observed is, at least, something outside of or in addition to mechanisms acting through breakdown of a pH gradient. While one would hesitate to use these data to justify a separate "transitional coupling" theory of uncoupler action, it is clear both from the specificity of the bilobe formation and from the similarity of the curves produced by four chemically dissimilar uncoupling compounds, that there is more going on during the process than expected from a straightforward induction of change in membrane proton permeability. Relevant to this are the ideas presented in recent reviews on the possibility of conformational coupling of ATP synthesis and electron transfer in the mitochondrial membrane (13, 14). As pointed out in those articles, because the energy involved in conformational changes is sufficient to drive phosphorylation and because these changes are "likely to be accompanied by .... movement of protonated groups," both the normal function and the disruption of energy coupling may be mechanistically complex, involving both control of proton movement and specific conformational alteration in respiratory chain proteins. The results presented here would seem to support the view that the mechanism of uncoupling, at least, involves conformational phenomena as well as those of proton translocation. 1. Reed, J., Reed, T. A. & Hess, B. (1978) Eur. J. Biochem. 91, 255-261. 2. Reed, J. & Hess, B. (1977) Hoppe-Seyler's Z. Physiol. Chem. 385, 1119-1124. 3. Urry, D. W. (1965) Proc. Natl. Acad. Sci. USA 54,640-648. 4. Urry, D. W. & van Gelder, B. F. (1968) in Structure and Functions of Cytochromes, eds. Okenuki, K., Kamen, M. B. & Sekuzo, I. (Univ. of Tokyo Press, Tokyo), pp. 210-214. 5. Urry, D. W. (1967) J. Am. Chem. Soc. 89,4190-4196. 6. Urry, D. W. & Pettegrew, J. W. (1967) J. Am. Chem. Soc. 89, 5276-5283. 7. Weiss, H. (1978) in Energy Conservation in Biological Membranes, 29th Mosbacher Colloquium (Springer, Heidelberg, West Germany), pp. 31-42. 8. Erecinska, M., Blasie, J. K. & Wilson, D. F. (1977) FEBS Lett.
76,235-240.
9. Perutz, M. F. (1970) Nature (London) 228,726-739. 10. Tiesjema, R. H. & van Gelder, B. F. (1974) Biochim. Biophys. Acta 347, 202-214. 11. Palmer, G., Babcock, G. T. & Vickery, L. E. (1976) Proc. Nati. Acad. Sci. USA 73,2206-2210. 12. Babcock, G. T., Vickery, L. E. & Palmer, G. (1976)J. Biol. Chem.
251,7907-7919. 13. Boyer, P. D. (1977) Annu. Rev. Biochem. 46,955-1026. 14. Slater, E. C. (1977) Annu. Rev. Biochem. 46, 1014-1026.
