ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 200, No. 2, April 1, pp. 387-395, 1980

Evidence that Cytochrome b Is the Antimycin-Binding Component of the Yeast Mitochondrial Cytochrome bc, Complex

HENRY ROBERTS, STUART C. SMITH, SANGKOT MARZUKI, AND ANTHONY W. LINNANE

Department of Biochemistry, Monash University, Clayton, Victoria, 3168, Australia

Received July 13, 19’79; revised October 23, 19’79

The binding of antimycin was studied in several mutant strains of yeast that have specific defects in cytochrome b. The strains have mutations in a part of the mitochondrial DNA that contains the structural gene for the apoprotein of cytochome b. Two of the mutants lack this protein and have no spectral cytochrome b. These mutants also lack the strong antimycin- binding site that is present in wild-type yeast mitochondria in the ratio of one site per two cytochrome b molecules. A third mutant which contains normal levels of spectral cytochrome b, but shows an altered absorption maximum for cytochrome b at 77”K, was found to bind normal amounts of antimycin. However, the fluorescence of antimycin bound to mito- chondria of this strain was found to be less efficiently quenched than in the case of the wild- type strain. In another mutant which contains only 20% of the normal spectral level of cyto- chrome b, the number of antimycin-binding sites was proportionately less. In an antimycin- resistant mutant, the binding of antimycin was too weak to be detected. The simultaneous modification of the structure of cytochrome b and the alteration of the antimycin-binding site in these mutants suggests that the antimycin-binding site is located on the apoprotein of cytochrome b.

Antimycin has profound effects on the function of QH,-cytochrome c reductase (for reviews, see Refs. (1, 2)). Because of the diversity of these effects, the antimycin- binding component must be considered as an important component in the structure of the complex, but it has not been identified, despite extensive investigations.

The main candidate for the antimycin- binding component is cytoehrome b, be- cause of its strong interaction with anti- mycin. The sigmoidal curves describing the relation between the inhibitory and spectral effects of antimycin on cytochrome b and the antimycin concentration have been interpreted in terms of allosteric binding of antimycin (3). From the quenching of the fluorescence of antimycin that occurs on binding to mitochondria or isolated Com- plex III, it has been calculated that the bound antimycin is within 20 A of the b heme (4). Two reports have suggested that the ratio of antimycin-binding sites to cyto- chrome b molecules remains constant (at 1:2), even when the amount of cytochrome

b is altered by partial purification (5) or by inhibition of mitochondrial protein synthe- sis (6). No such relationship exists between the binding of antimycin and the concentra- tion of cytochrome c, (5) or iron-sulfur protein (‘7). The isolation of antimycin- resistant mutants of yeast (8-10) that con- tain mutations mapping in the region of the mitochondrial DNA coding for the struc- tural gene for cytochrome b (ll- 15) sug- gests that the antimycin-binding site is on the cytochrome b protein.

However, there is no direct evidence that cytochrome b binds antimycin. On the con- trary, Das Gupta and Rieske (16) have ten- tatively suggested that antimycin binds, not to cytochrome b but to a previously un- characterized “antimycin-binding protein.” By photoaffinity labeling of Complex III using an analog of antimycin, a protein having a molecular weight of 11,500 was specifically labeled. The authors later ac- cepted this result with reservations (2), and it cannot be ruled out that the labeled com- ponent was a contaminant or even a product

387 0003-9861/80/04038’7-09$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved

388 ROBERTS ET AL.

of proteolysis of cytochrome 6. Direct bind- ing studies with isolated cytochrome b are not feasible at present, since its chemical properties are modified during purification (17, 18). Modified cytochrome b does not bind antimycin (5).

A class of respiratory-deficient yeast mutants having specific defects in cyto- chrome b (cyb mutants) is now available in this laboratory (8, 11, 19). In this report, alterations to the binding of antimycin are correlated with the modifications of the cytochrome b apoprotein in these strains, providing further evidence that antimycin binds to cytochrome b.

MATERIALS AND METHODS

Materials. Antimycin, cholic acid, and the three protease inhibitors phenylmethylsulfonyl fluoride, E- aminocaproic acid and p-aminobenzamidine-HCl, were obtained from Sigma Chemical Company (St. Louis, MO.). Bovine serum albumin was from Commonwealth Serum Laboratories (Melbourne). SDS was obtained from Pierce (Rockford, Ill.). Acrylamide (Eastman Kodak Co., Rochester, N. Y.) and N,N’-methylene- bisacrylamide (Bio-Rad, Richmond, Calif.) were purified using an ion exchange resin (Bio-Rad AG 501-X8(D)). N,N,N’,N’-Tetramethylethylenedia- mine and ammonium persulfate were obtained from BDH Chemicals Ltd. (Poole, England).

Yeast strains, growth conditions, and isolation of mitochondria. The wild-type strain of Sacchuromyces cereuisiae used was J69-1B (a ade his). The cyb

mutants 17-62-2, 37-16-6, 41-2-4, and 1505, and the antimycin-resistant mutant A812 were derived from it, and have been shown to contain mutations in the mito- chondrial DNA within the region between 22 and 25 map units ((19), and R. M. Hall, unpublished).

Strains J69-1B and A812 were grown in batch cul- ture and harvested in the midlogarithmic phase (20). The respiratory-deficient cyb mutants were grown under derepressed conditions in a glucose-limited chemostat (21). Cells were suspended in 13 mM Tris- HCl (pH 7.4) containing 0.33 M mannitol, 0.27 M

sorbitol, and 0.7 mM EDTA (resuspension buffer). They were disrupted by shaking with glass beads for 30 s, and mitochondria were isolated from them, es- sentially as described previously (20).

Labeling and SDS-polyacrylumide gel analysis.

Mitochondrial translation products were labeled with 35S as described previously (22). Mitochondria were then isolated from the labeled cells as above, in the presence of the protease inhibitors phenylmethyl- sulfonyl fluoride (0.5 mM), p-aminobenzamidine (5

’ Abbreviations used: SDS, sodium dodecyl sulfate.

mrv0, and e-aminocaproic acid (5 mM). Aliquots of the suspensions, containing equal amounts of radioac- tivity, were treated at 100°C for 2 min with SDS (2% final concentration), sucrose (140/o), Tris (0.12 M, pH 6.7), 0.025% bromphenol blue, 2-mercaptoethanol (l%), and protease inhibitors as above, in a final volume of 50 hl. After cooling, the samples were analyzed by electrophoresis on SDS-polyacrylamide gels as de- scribed by Studier (23) and LaemmIi and Favre (24). After staining the gels, the labeled bands were de- tected by scintillation autoradiography (25).

Antimycin-binding to mitochondtia. The binding of antimycin to mitochondria was assayed by the method of Berden and Slater (4). Antimycin, dissolved in 20 ~1 of methanol, was added to mitochondria (1 mg pro- tein) suspended in resuspension buffer containing 6 mg/ ml bovine serum albumin. After 20 min at room tem- perature, the samples were centrifuged for 12 min at 17,500g (maximum). An aliquot of the supernatant was diluted to 3 ml with the above buffer for fluores- cence measurements.

Spectral measurements. All spectral measurements were made with an Aminco DW-2a spectrophotom- eter, with attachments for recording spectra at 77°K and total fluorescence intensities. Antimycin fluorescence was excited at 355 nm with a 20-nm bandpass through a filter cutting off above 410 nm, and the emission was cut off below 410 nm with a Wratten ZA filter. For low-temperature spectra, mitochondria were taken up in resuspension buffer and were reduced or oxidized as described in the figure legends, before freezing. The sample holder had a nominal path length of 2 mm.

Assay methods. Antimycin concentrations were de- termined using the published extinction coefficient of 4.8 mM-* cm-* (320 nm; ethanol) (26). Protein was determined by the method of Lowry et al. (27).

RESULTS

Biochemical Properties of cyb Mutants

Four strains, 17-62-2, 37-16-6, 41-2-4, and 1505, which carry mitochondrial muta- tions affecting cytochrome b, and the parental strain J69-lB, were selected in order to determine whether different modi- fications of cytochrome b influence the bind- ing of antimycin. Three of these mutants have previously been characterized both genetically and biochemically in this labora- tory (11, 19). The biochemical properties of the strains are summarized in Table I. Mitochondria isolated from strains 17-62-2 and 41-2-4 lack spectrally detectable cyto- chrome b, and have very low NADH: cytochrome c reductase activities. The

BINDING OF ANTIMYCIN TO CYTOCHROME b 389

TABLE I

CYTOCHROME CONTENTANDENZY~EACTIVITIESOFMITOCHONDRIAISOLATEDFROM cyb MUTANTS

Strain

Mitochondrial cytochromes” (nmol heme/mg protein)

b aa3 c +c,

Enzyme activitiesb (pmollmin ‘rng protein)

NADH:cytochrome Cytochrome c reductase oxidase ATPase’

J69-1B 0.29 17-62-2 Trace 41-2-4 Trace 37-16-6 0.31 1505 0.07

0.31 0.42 0.46 0.42 1.26(92) 0.44 0.44 0.01 0.16 1.26(94) 0.20 0.28 0.01 0.10 1.12(>95) 0.48 0.52 0.05 0.20 1.17(93) 0.06 0.25 0.035 0.27 0.77(>95)

a The cytochrome content was determined using published reduced-oxidized extinction coefficients (5,28,29). b Enzyme activities were assayed by the following procedures: antimycin-sensitive NADH:cytochrome c

reductase (30), cytochrome oxidase (31), and oligomycin-sensitive ATPase (32). c Percentage inhibition of ATPase activity by oligomycin is given in parentheses.

levels of the other cytochromes are com- parable to those of the parental strain J69-1B. Strain 3’7-16-6 contains normal levels of all cytochromes but the absorp- tion maximum of cytochrome b at 77°K is red-shifted by 2.5 nm compared with that of the parent J69-1B (Ref. (11) and Fig. 1). The NADH:cytochrome c reductase ac- tivity of mitochondria of strain 37-16-6 is 15% of that of strain J69-lB, and is only 60% inhibitable by antimycin. Strain 1505 has only about 20% of the spectral cyto- chrome b content of the wild type, and the NADH:cytochrome c reductase activity is less than 10% of the wild-type level. All of the mutants show significant cytochrome oxidase activities, although these are con- sistently lower than that of the wild type. The ATPase activities of the mutants are comparable to that of the wild type, and are fully sensitive to oligomycin.

Low-Temperature Spectra of cyb Mutants

The spectral components of mitochondria isolated from the parental and mutant strains were examined in detail at 77°K. Figure 1 shows that the cytochrome b ab- sorption band, at 559 nm in the parental strain, is considerably less intense in strain 1505, and is absent in strains 17-62-2 and 41- 2-4. The slight shoulder at 559 nm in the lat- ter strains is not regarded as being due to traces of mitochondrial cytochrome b, since

it has also been observed in mitochondria from petite mutants of yeast, which con- tain no mitochondrially synthesized cyto- chromes (H. Roberts and K. K. Mahesh- wari, unpublished).

In mitochondria of strain 37-16-6 reduced with dithionite (Fig. l), the cytochrome b band has a maximum at 561.5 nm, but may contain a second component which appears as a slight shoulder at about 560 nm. In succinate-reduced mitochondria of this strain (Fig. 2, trace 3), the cytochrome b band is at 560.5 nm, compared with 559 nm in the parental strain J69-1B (Fig. 2, trace 1). Thus both the succinate-reducible com- ponent and the component that cannot be reduced by succinate are shifted to longer wavelengths in this mutant. In the presence of antimycin, the cytochrome b band is in- tensified and shifted to the red, although the extent of the shift in the mutant (Fig. 2, trace 4), is less than in the parental strain (trace 2).

It is difficult to quantify the cytochrome c1 content of the mitochondria from these spectra, due to the overlap with other bands and the uncertainty in the degree of in- tensification at 77°K. The absorption band of cytochrome c,, at 553.5 nm in strain J69- lB, is present in all four mutants, although only as a shoulder in strain 41-2-4. How- ever, the absence of the cytochrome b band in this strain may influence the apparent in- tensity of the cytochrome c, band. It is con-

390 ROBERTS ET AL.

I

560 560 600 Wavelength (nm)

FIG. 1. Low-temperature difference absorption spectra of mitochondria from parental and cyb mutant strains. The protein concentrations of the mitochondria were as follows: J69-1B (2.43 mg/ml); 37-16-6 (0.79 mg/ml); 1505 (1.54 mg/ml); 17-62-2 (0.59 mg/ml); and 41-2-4 (0.46 mg/mI). The sample suspensions were reduced with a few grains of sodium dithionite, and the references were oxidized with a crystal of am- monium persulfate. The calibration bar represents 0.002 A for strain 17-62-2, and 0.005 A for the other strains.

eluded that cytochrome c1 is incorporated into the mitochondria of the mutants, but possibly to a lesser extent in strain 41-2-4.

Analysis of Mitochondrially Translated Polypeptides in cyb Mutants

The apoprotein of cytochrome b, which has a molecular weight of approximately 32,000 (33, 34), is the only product of mito- chondrial protein synthesis that is asso- ciated with the cytochrome bc, complex (35). In order to determine whether muta- tions in the cyb locus affect the struc- ture or the synthesis of apocytochrome b,

the mitochondrial translation products were labeled with 35S and the isolated mito- chondria were analyzed by electrophoresis on polyacrylamide slab gels in the presence of SDS. Scintillation autoradiographs of the gels of wild-type mitochondria (Fig. 3) show seven major labeled bands, of which the band at the position corresponding to a molecular weight of 32,000 is attributed to the apoprotein of cytochrome b (12,1’7). This band is also present in strains 1505 and 37-16-6. The spectral properties of cyto- chrome b are specifically modified in these mutants (Table I and Fig. l), suggesting that the mutations affect the structure of cytochrome b. However, the alterations of the apoprotein of cytochrome b appear to be

succinale

37-16-6 + succinaie

I40 560 580 Wavelength (nm)

FIG. 2. Low-temperature spectra of mitochondria reduced with succinate. The protein concentrations of the mitochondria were 1.2 mg/ml for J69-1B and 1.4 mg/ml for 37-16-6. In traces 1 and 3, the sample was reduced with 60 mM succinate and incubated at room temperature for 15 min to allow most of the oxygen to be consumed, before freezing. In traces 2 and 4, anti- mycin (1.5 pg/ml) was added to the sample 2 min be- fore the succinate. In all cases the reference con- tained air-oxidized mitochondria.

BINDING OF ANTIMYCIN TO CYTOCHROME b 391

too small to affect its electrophoretic mobility in these gels. In strains 17-62-2 and 41-2-4 the band corresponding to apo- cytochrome b is absent, suggesting that the mutations prevent the synthesis of the apo- protein. Although the intensity of the band due to the proteolipid subunit of the ATPase (M, 7,600) is decreased in strains 41-2-4 and 17-62-2, the oligomer of this protein (M, 50,000) is present in these strains.

Quenching of Antimycin Fluorescence on Binding to Mitochondria

The interaction of antimycin with mito- chondria was studied by observing the quenching of the fluorescence of antimycin, due to energy transfer to the heme group of cytochrome b (4). As shown in Fig. 4a, mito- chondria of the wild-type strain J69-1B quench the fluorescence of antimycin at low concentrations of antimycin. Subsequently, there is a steep increase in the fluores- cence, after which the curve becomes parallel to that observed in the absence of mitochondria. The biphasic changes in the fluorescence indicate that a combination of two effects is occurring. The initial quench- ing of the fluorescence is presumably due

a b c d e Standards Wr)

-45.ow

CYtochrc.ne b -4o.ow

armprotein -- -25.000

-12.500

FIG. 3. SDS-polyacrylamide gel electrophoresis of mitochondrial translation products of wild-type and cyb

mutants. The samples, prepared as described under Materials and Methods, were loaded onto a 12.5% poly- acrylamide gel, together with the following standards: bovine serum albumin, ovalbumin, aldolase, chymo- trypsinogen, and cytochrome c. After electrophoresis the gel was stained with Coomassie blue and the labeled bands were detected by scintillation auto- radiography (25). Samples a, b, c, d, and e contained mitochondria from strains J691B, 1505, 3’7-16-6, 41- 2-4, and 1’7-62-2, respectively.

2 rnmycm

FIG. 4. Quenching of antimycin fluorescence by mito- chondria in the absence of bovine serum albumin. Aliquots (2 ~1) of antimycin A in methanol were added to 3 ml of resuspension buffer (0) or 3 ml of re- suspension buffer + mitochondria (0). (a) J69-1B (8.2 mg protein; 2.1 nmol cytochrome b). (b) 17-62-2 (1.17 mg protein). (c) 41-2-4 (5.5 mg protein). (d) 37-16-6 (7.1 mg protein; 2.2 nmol cytochrome b).

to the binding of antimycin at a site close to the b heme. This site is saturated by the equivalent of about 0.4 mol of antimycin/ mol of cytochrome b.

The reason for the steep increase of the fluorescence above this point is not clear. One possible explanation is that the reduc- tion of cytochrome b by endogenous sub- strates, due to inhibition of electron trans- fer by antimycin, becomes significant in this region of the titration (data not shown). Reduced cytochrome b is known to bind antimycin less lirmly and to quench the fluorescence less efficiently than does oxidized cytochrome b (4). However, the increase of the fluorescence seems too great to be accounted for by this effect, unless it is proposed that reduced cytochrome b ac- tually enhances the fluorescence of the bound antimycin, which is not the case in Complex III from beef heart (4).

Another reason for the fluorescence in- crease may be antimycin binding to a second, weaker site where its fluorescence is enhanced. The number of such binding sites, estimated from the concentration range over which the increase occurs, would

392 ROBERTS ET AL.

FIG. 5. Antimycin fluorescence titration of mito- chondria in the presence of bovine serum albumin. Aliquots (2 ~1) of antimycin in methanol were added to 3 ml of resuspension buffer + bovine serum al- bumin (6 mg/ml) (0) or 3 ml of resuspension buffer + bovine serum albumin (6 mg/ml) + mitochondria (0). (a) J69-1B (8.2 mg protein; 2.1 nmol cyto- chrome 5). (b) 17-62-2 (5.9 mg protein). (c) 41-2-4 (1.0 mg protein). (d) 37-16-6 (7.9 mg protein; 2.4 nmol cyto- chrome 5). (e) 1505 (15.4 mg protein; 1.0 nmol cyto- chrome 5). (f) A812 (6.8 mg protein; 2.0 nmol cyto- chrome 5).

be about 0.25 mol/mol of cytochrome b, or about half of the number of stronger bind- ing sites. Burger and co-workers (36) have reported that there are two antimycin- binding sites in mitochondria of the yeast Schixosaccharomyces pombe, but they did not determine whether the fluorescence of antimycin was quenched or enhanced on binding.

In contrast to the wild type, mitochondria of the mutants 17-62-2 and 41-4-2 do not quench or enhance the fluorescence of anti- mycin (Figs. 4b, c). In some cases the fluorescence intensities are slightly less than in the absence of mitochondria, due to absorption of the exciting and emitted light by mitochondria. Since the mutants

17-62-2 and 41-2-4 contain no spectral cyto- chrome b, it would be expected that the fluorescence of antimycin bound to the mutant mitochondria would be enhanced, as is the case when antimycin binds to bovine serum albumin (37). The lack of any fluo- rescence enhancement indicates that there are no strong antimycin-binding sites in these mutants.

Mitochondria of strain 37-16-6 produce a slight quenching of the fluorescence at antimycin levels below 0.5 mol/mol of cyto- chrome b (Fig. 4d). These results sug- gest that there is some binding of antimy- tin in this mutant, but the altered cyto- chrome b quenches the fluorescence of bound antimycin less efficiently than normal cytochrome b.

Binding of Antimycin to Mitochondria

The binding of antimycin was assayed by two methods. In the first, mitochondria were titrated with antimycin in the presence of bovine serum albumin, which increases the fluorescence of the free antimycin several fold (4). Binding is therefore ob- served as a decrease in fluorescence. Figure 5a shows that antimycin is bound stoichio- metrically up to 0.5 mol/mol of cytochrome b in strain J69-1B. The later increase of the fluorescence observed when the titra- tion was carried out in the absence of bovine serum albumin (Fig. 4a) is not seen in the presence of bovine serum albumin. The reason for this is probably that the enhance- ment of the fluorescence of antimycin bound to bovine serum albumin masks any en- hancement on binding to mitochondria.

Strains 17-62-2 and 41-2-4 do not bind antimycin (Figs. 5b, c). Similarly, some other cyb mutants containing no spectral cytochrome b were found not to bind anti- mycin (data not shown). Mitochondria of strains 37-16-6 and 1505 bind 0.5 mol of anti- mycinlmol of cytochrome b (Figs. 5d, e) as for wild-type mitochondria, although both the number of binding sites per milligram of protein and the cytochrome b content in strain 1505 are only about 20% of those of the wild type. No binding of antimycin could be detected in mitochondria of the anti- mycin-resistant strain A812 (Fig. 5f).

BINDING OF ANTIMYCIN TO CYTOCHROME b 393

Identical results were obtained with three other antimycin-resistant strains (data not shown). Inspection shows that the binding of antimycin would be undetectable by this method if the dissociation constant were more than loo-fold greater than in wild- type mitochondria. In contrast to the cyb mutants, the antimycin-resistant strains contain fully functional mitochondria, with identical low-temperature spectra to the wild-type (data not shown).

In the second method of assaying the binding, the mitochondria were centrifuged and the fluorescence of the free antimycin was measured (Fig. 6). With strains J69- 1B and 37-16-6, little fluorescence appears in the supernatant until the amount of anti- mycin present exceeds 0.5 mol/mol of cyto- chrome b. Above this point, the fluorescence increases linearly, but the slope is less than in the absence of mitochondria, suggesting that there are some weaker, nonspecific binding sites.

With strains 17-62-2 and 41-2-4, the fluorescence increases linearly over the whole range of the titration, indicating that there are no strong antimycin-binding sites. However, the fluorescence increases less steeply than in the absence of mitochondria, in strain 17-62-2, suggesting weak, non- specific binding to the mitochondria, as in strain J69-1B.

DISCUSSION

These studies clearly show that the specific antimycin-binding site that is pre- sent in the ratio of one site per two mole- cules of cytochrome b in wild-type mito- chondria (Figs. 5 and 6) is absent in mito- chondria of the two mutants (17-62-2 and 41-2-4) which lack spectral cytochrome b. The mutations in these strains appear to prevent the synthesis of apocytochrome b. In strain 1505, the apoprotein of cyto- chrome b appears normal, but the spec- trum and enzymic activity of the mito- chondria indicate that they contain much reduced levels of properly assembled cyto- chrome b. However, the ratio of the number of antimycin-binding sites to the number of spectrally detectable cytochrome b mole- cules remains 1:2, suggesting that only the

FIG. 6. The binding of antimycin to mitochondria. Individual samples containing 1 mg of mitochondria (0) or no mitochondria (0) were treated as described under Materials and Methods, and the fluorescence of the free antimycin was measured. (a) J69-1B (0.31 nmol cytochrome b). (b) 17-62-2. (c) 41-2-4. (d) 37- 16-6 (0.30 nmol cytochrome b).

small amount of cytochrome b which is properly assembled binds antimycin in the normal way. In strain 37-16-6, the altered cytochrome b spectrum indicates that the mutation affects the environment of the heme group, and the less efficient quenching of antimycin fluorescence indicates that the orientation or position of the bound anti- mycin relative to the b heme group is altered.

Further evidence that the mutational al- teration of cytochrome b modifies the anti- mycin-binding site was obtained with the antimycin-resistant mutants. These strains carry mutations in the same region of the mitochondrial DNA as those in strains 17- 62-2, 37-16-6, 41-2-4 and 1505 ((15), and R. M. Hall, unpublished). The binding of antimycin to mitochondria of the antimy- cm-resistant strains was undetectable in the presence of bovine serum albumin, showing that the binding is much weaker than in the parental strain.

It is possible that the loss of antimycin- binding sites in mitochondria of strains 17- 62-2, 41-2-4, 1505, and A812 is a secondary result of pleiotropic effects of the muta- tions on other membrane components. However, this is unlikely, because such pleiotropic effects do not seem to be very

394 ROBERTS ET AL,

extensive and do not occur in all of the mutants. Thus, the amount of the mono- meric form of the proteolipid subunit of the ATPase in strains 17-62-2 and 41-2-4 is slightly lower than in the wild type (Fig. 3), but the oligomeric form is present in normal amounts, and the ATPase activity and its sensitivity to oligomycin are not signifi- cantly decreased (Table I). The amount of cytochrome oxidase subunit I (M, 44,000) also varies somewhat between the different strains (Fig. 3), but this variation bears no relationship to the number of antimycin- binding sites.

REFERENCES

SLATER, E. C. (1973) Biochim. Biophys. Acta 301, 129- 154.

RIESKE, J. S. (1976) Biochim. Biophys. Acta

456, 195-247.

On the other hand, there is a strong correlation between modifications of cyto- chrome b and of the antimycin-binding site, which supports the previous indirect evidence implicating cytochrome b as the antimycin-binding component of the cyto- chrome bc, complex. Many studies have previously shown that there are two func- tionally distinct forms of cytochrome b (for review see Ref. (2)). It has been proposed that antimycin binds only to the long-wave- length form of cytochrome b(“bT”) (6, 38). Such hypotheses would account for the fact that there are two molecules of cytochrome b per antimycin-binding site, but are in- consistent with the fact that antimycin af- fects both species of cytochrome b. It is not yet clear whether the spectrally distin- guishable forms of cytochrome b represent different molecular species or a single species in different environments. Weiss (17) has suggested that cytochrome b from Neurospora crassa consists of two slightly different polypeptide chains, but it is not known whether they are the apoproteins of spectrally different cytochromes b. The ob- servation (Figs. 1 and 2) that a single muta- tion in strain 37-16-6 affects both the ab- sorption spectrum of the cytochrome b species reduced by succinate, and that of the total cytochrome b reduced by dithionite or by succinate in the presence of antimycin, suggests that the two types of cytochrome b are products of a single mitochondrial gene.

1.

2.

3.

4.

5.

6.

7.

8.

BRYLA, J., KANIUGA, Z., AND SLATER, E. C. (1969) Biochim. Biophys. Acta 189, 317-326.

BERDEN, J. A., AND SLATER, E. C. (1972) Bio-

chim. Biophys. Acta 256, 199-215. BERDEN, J. A., AND SLATER, E. C. (1970)

Biochim. Biophys. Acta 216,237-249.

VON JAGOW, G., AND KLINGENBERG, M. (1972) FEBS Lett. 24, 278-282.

RIESKE, J. S., ZAUGG, W. S., AND HANSEN, R. E. (1964) J. Biol. Chem. 239, 3023-3030.

GROOT OBBINK, D. J., HALL, R. M., LINNANE, A. W., LUKINS, H. B., MONK, B. C., SPITHILL,

T. W., AND TREMBATH, M. K. (1976) in The Genetic Function of Mitochondrial DNA (Sac-

cone, C., and Kroon, A. M., eds.), pp. 163-173, Elsevier, Amsterdam.

9.

10.

BURGER, G., LANG, B., BANDLOW, W., SCHWE- YEN, R. J., BACKHAUS, B., AND KAUDEWITZ,

F. (1976) Biochem. Biophys. Res. Commun. 72, 1201-1208.

MICHAELIS, G. (1976) Mol. Gen. Genet. 146,

133- 137.

11. COBON, G. S., GROOTOBBINK, D. J., HALL, R. M., MAXWELL, R., MURPHY, M., RYTKA, J., AND

LINNANE, A. W. (1976) in Genetics and Bio- genesis of Chloroplasts and Mitochondria (Bucher, T., Neupert, W., Sebald, W., and

Werner, S., eds.) pp. 453-459, Elsevier, Amsterdam.

12. TZAGOLOFF, A., FOURY, F., AND AKAI, A. (1976) Mol. Gen. Genet. 149, 33-42.

13. COLSON, A. M., AND SLONIMSKI, P. P. (1977) in Mitochondria 1977. Genetics and Biogenesis of

Mitochondria (Bandlow, W., Schweyen, R. J., Wolf, K., and Kaudewitz, F., eds.), pp. 185- 198, de Gruyter, Berlin.

14. CLAISSE, M. L., SPYRIDAKIS, A., AND SLONIM- SKI, P. P. (1977) in Mitochondria 1977. Genetics

and Biogenesis of Mitochondria (Bandlow, W., Schweyen, R. J., Wolf, K., and Kaudewitz, F.,

eds.), pp. 337-344, de Gruyter, Berlin.

15. LINNANE, A. W., AND HALL, R. M. (1978) in Molecular Biology of Mitochondrial Membranes

(Fleischer, S., Hatefi, Y., MacLennan, D. H., and Tzagoloff, A., eds.), pp. 321-335, Plenum, New York.

ACKNOWLEDGMENTS

16. DAS GUPTA, U., AND RIESKE, J. S. (1973) Bio- them. Biophys. Res. Commun. 54.1247-1254.

17. WEISS, H. (1976) B&him. Biophys. Acta 456, 291-313.

The authors wish to thank Dr. G. K. Radda for 18. LIN, L.-F. H., AND BEATTIE, D. S. (1978) J. stimulating discussions and suggestions. We are grate- Biol. Chem. 253, 2412-2418.

ful to Miss N. Mikhail for technical assistance. 19. RYTKA, J., ENGLISH, K., HALL, R. M., LINNANE,

BINDING OF ANTIMYCIN TO CYTOCHROME b 395

A. W., AND LUKINS, H. B. (1976) in Genetics

and Biogenesis of Chloroplasts and Mito-

chondria(Bucher, T., Neupert, W., Sebald, W.,

and Werner, S., eds.), pp. 427-434, Elsevier, Amsterdam.

20. ROBERTS, H., CHOO, W. M., SMITH, S. C.,

MARZUKI, S., LINNANE, A. W., PORTER, T. H., AND FOLKERS, K. (1978) Arch. Biochem. Biophys. 191, 306-315.

21. MARZUKI, S., COBON, G. S., HASLAM, J. M., AND LINNANE, A. W. (1975) Arch. Biochem.

Biophys. 169,577-590. 22. MURPHY, M., GIJTOWSKI, S. J., MARZUKI, S.,

AND LINNANE, A. W. (1978) Biochem. Biophys.

Res. Commun. 85, 1283-1290.

23. STUDIER, F. W. (1973) J. Mol. Biol. 79, 237- 248.

24. LAEMMLI, U. K., AND FAVRE, M. (1973) J. Mol. Biol. 80, 575-599.

25. BONNER, W. M., AND LASKEY, R. A. (1974) Eur. J. Biocbm. 46, 83-88.

26. STRONG, F. M., DICKIE, J. P., LOOMANS, M. E., VANTAMELEN, E. E., ANDDEWEY, R. S. (1960)

J. Amer. Chem. Sot. 82, 1513-1514. 27. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.,

AND RANDALL, R. J. (1951) J. Biol. Chem. 193,

265-275.

28. VAN GELDER, B. F., AND SLATER, E. C. (1963)

Biochim. Biophys. Acta 73, 663-665. 29. VANNESTE, W. H. (1966) Biochim. Biophys. Acta

113, 175-178.

30. HATEFI, Y., AND RIESKE, J. S. (1967)in Methods in Enzymology (Estabrook, R. W., and Pull- man, M. E., eds.), Vol. 10, pp. 225-231,

Academic Press, New York.

31. WHARTON, D. C., AND TZAGOLOFF, A. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 245-250,

Academic Press, New York.

32. COBON, G. S., AND HASLAM, J. M. (1973) Bio- them. Biophys. Res. Commun. 52, 320-326.

33. WEISS, H., AND ZIGANKE, B. (1974) Eur. J. Bio- chm. 41, 63-71.

34. KATAN, M. B., POOL, L., AND GROOT, G. S. P.

(1976) Eur. J. Biochem. 69, 95-105. 35. KATAN, M. B., VAN HARTEN-LOOSBROEK, N.,

AND GROOT, G. S. P. (1976) Eur. J. Biochem. 70, 409-417.

36. BURGER, B., LANG, B., BANDLOW, W., AND KAUDEWITZ, F. (1975) Biochim. Biophys. Acta 396, 187-201.

37. REPORTER, M. (1966) Biochemistry 5,2416-2423.

38. STOREY, B. T. (1972) Biochim. Biophys. Acta 267, 48-64.