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[13] Characterization and resolution of complex III from beef heart mitochondria
80
[13]
ELECTRON TRANSFER COMPLEXES
[13] C h a r a c t e r i z a t i o n and Resolution of Complex from Beef Heart Mitochondria
By
III
B . D E A N NELSON a n d P,~R GELLERFORS
Peptide Composition of Complex III Peptide Composition of Complex III as Determined by Sodium Dodecyl Sulfate(SDS)-Gel Electrophoresis
Electrophoretic Procedures Complex III is dissolved in a mixture of 2% SDS, 5% 2-mercaptoethanol, and 10 mM Tris-chloride, pH 6.8, to a final concentration of 1-2 mg of protein per milliliter. The solution is incubated for 3 rain at 100°, and 20-50 tzg of protein are applied to 12.5% (w/v) polyacrylamide gels containing a constant ratio of acrylamide to methylene bisacrylamide of 37 : 1. Preparation of the gels and electrophoresis are carried out according to the methods of Laemmli.l
Major Peptides of Complex Ili Complex III z contains seven major peptides (Fig. 1A), the molecular weights of which are approximately 50,000, 47,000, 29,000, 26,000, 15,000, 13,000, and 10,000 when separated by electrophoresis as described above. The molecular weight of 10,000 for the last peptide appears to be overestimated in this system. In more highly cross-linked gels, this band can be resolved into several peptides with molecular weights less than 10,000. a.5 The identities of the above peptides are far from certain, and in very few cases can a function be absolutely associated with each fraction. Furthermore, rigorous analysis showing peptide homogeneity within each band has not been accomplished. On the other hand, all complex III preparations so far reported from beef heart 3-11 or yeast TM mitochondria i U. K. Laemmli, Nature (London) 227, 680 (1970). 2 j. S. Rieske, W. S. Zaugg, and R. E. Hansen, J. Biol. Chem. 239, 3023 (1964). :t C. A. M. Marres and E. C. Slater, Biochim. Biophys. Acta 462, 531 (1977). 4 p. Gellerfors and B. D. Nelson, Eur. J. Biochem. 52, 433 (1975). 5 R. L. Bell and R. A. Capaldi, Biochemistry 15, 996 (1976). 6 H. Baum, H. I. Silman, J. S. Rieske, and S. H. Lipton, J. Biol. Chem. 242, 4876 (1967). 7 U. D. Gupta, and J. S. Rieske, Biochem. Biophys. Res. Commun. 54, 1247 (1973). 8 j. F. Hare and F. L. Crane, Sub-Cell. Biochem. 3, 1 (1974).
[13]
81
COMPLEX 11t FROM BEEF HEART
A
B
C
D
E
F
G
H
50,000 47,000 29,000 26,000
"
W
o ~!iiii~/
~
i~ii!i~I
15,000 13,000 10,000
FIG. I. Separation of the peptides of complex 111. Separation of the peptides and sodium dodecyl sulfate (SDS)-gel electrophoresis were carried out as described in the text. A, Complex lIl: B, cytochrome b fraction after splitting the complex; C, cytochrome cl fraction after splitting the complex; D, core protein; E, cytochrome cl heme-binding peptide; F, supernatant after removal of cytochrome c, heine-binding peptide: G, iron-sulfur protein; H, cytochrome b fraction.
have a characteristic peptide pattern similar to that shown in Fig. 1A with respect to the relative positions of the peptides on the gels and to their relative staining intensities. The major peptides listed above probably represent the basic peptide unit of complex III. With the above reservations, the major peptides can be given an assignment based upon the enrichment or isolation of their prosthetic groups. The molecular weight (M0 estimates differ slightly in various laboratories,3-~, 7-11,13 but the peptides can be identified by their charac9 C. A. Yu, L. Yu, and T. E. King, J. Biol. Chem. 249, 4905 (1974). lo R. A. Capaldi, Arch. Biochem. Biophys. 163, 99 (1974). u E. C. Slater, in "Electron Transfer Chains and Oxidative Phosphorylation" (E. Quagliariello, s. Papa, F. Palmieri, E. C. Slater, and N. Siliprandi, eds.), p. 3. North-Holland Publ., Amsterdam, 1975. 12 M. B. Katan, L. Pool, and G. S. P. Groot, Eur. J. Biochem. 65, 95 (1976). 13 R. A. Capaldi, R. L. Bell, and T. Branchek, Biochem. Biophys. Res. Commun. 74, 425 (1977).
82
ELECTRON TRANSFER COMPLEXES
[13]
teristic distributions on the gels and their staining intensities. Core proteins 1 and 2 (Mr 50,000 and 47,000) contain no prosthetic groups 14 but appear to be normal constituents of complex III, since their removal leads to loss of antimycin binding, ~ and since specific alkylation of core protein-1 with iodoacetamide leads to inactivation of enzyme activity.lG The Mr 29,000 peptide contains cytochrome ¢1 heme-binding peptide, which, because of the covalently bound heme, can be located directly on the gels. It has been suggested that this fraction, in analogy with Neurospora,~7 also contains cytochrome b. 3 The Mr 26,000 peptide corresponds to the isolated iron-sulfur protein. 2-4' s-10,18 The lower part of the gel contains 3 major peptides whose functions are not known and the identities of which have not been completely established. The smallest of these (Mr 10,000) has been associated with the binding of antimycin. 7 This band contains, however, several polypeptides with molecular weights of less than 10,000. 3"~ The remaining peptides (Mr 15,000 and 13,000) have been associated with cytochromes b or cl. Some confusion has arisen regarding these latter peptides, since they can switch positions on a gel, depending upon the electrophoretic system used.13 Additional peptides can also appear on the gels, especially in the area above Mr 60,000. They never make up more than 2% of the stainable protein and are often missing from the gels. They probably represent contaminations. Among the most prominent of these is a peptide of Mr 70,000, which is either the large subunit of succinate dehydrogenase 19 or an artifact of the electrophoretic system. 4 Peptides previously reported 4 in the molecular weight ranges of 17,000-18,000 and 34,000-37,000 are seldom observed as long as the samples are prepared for electrophoresis and the electrophoresis is carried out according to the procedures outlined above. The peptide pattern on SDS-gel is altered only slightly when the samples are prepared and electrophoresed in the absence of mercaptoethanol, indicating the absence of intermolecular disulfide bands. SDSgels stained for carbohydrate using periodic acid-Schiff reagent (PAS) show no positive staining reactions associated with the peptide bands. A positive reaction is observed near the dye front and probably represents carbohydrate-containing lipids. Molar ratios of the peptides have been estimated 3"4 to be: 50,000 (1 14 H. I. Silman, J. S. Rieske, S. H. Lipton, and H. B a u m , J. Biol. Chem. 242, 4867 (1967). 15 j. S. Rieske, S. H. Lipton, H. B a u m , and H. I. Silman, J. Biol. Chem. 242, 4888 (1967). 16 p. Gellerfors, M. L u n d 6 n , and B. D. Nelson, Eur. J. Biochem. 67, 463 (1976). lr H. Weiss, Biochim. Biophys. Acta 456, 291 (1976). 18 j. S. Rieske, D. H. M a c L e n n a n , and R. Colman, Biochem. Biophys. Res. Commun. 15, 338 (1964). 19 K. A. Davis, and Y. Hatefi, Biochemistry 10, 2509 (1971).
[13]
COMPLEXill FROMBEEF HEART
83
mol), 47,000 (1 mol), 29,000 (1 tool), 26,000 (1-2 mol), 15,000 (1 mol), 13,000 (2 mol), and 10,000 (2-6 tool). Isolation of the Peptides of Complex III Reagents 1. Sodium taurocholate (DIFCO), a 30% (w/v)solution in water 2. Ammonium sulfate, saturated at 00-4 ° and neutralized with ammonia 3. Imidazole, 1 M solution buffered to pH 6.6 with sulfuric acid 4. Antimycin, 1 mg/ml in ethanol 5. Sodium mersalyl, 50 mM solution in water 6. Sodium dodecyl sulfate (SDS), 10% (w/v) in water 7. Potassium cholate, recrystallized in 70% ethanol, 20% (w/v) in water Isolation Scheme Step 1. Separation of the Cytochrome b and Cytochrome c, fractions. 2°'21 To 100/xl of complex III (3 mg of protein) suspended in 0.66 M sucrose and 50 mM Tris-chloride, pH 8.0, is added 10/xl of imidazolesulfate solution followed by 110 tzl of water and 120 /zl of saturated ammonium sulfate. Separation of the cytochromes is initiated by addition of 60 p,1 of the taurocholate solution. After addition of taurocholate, the mixture is taken to 25 ° and incubated for 3 hr. The precipitate which forms contains 80-100% of the cytochrome b, and the supernatant contains 80-100% of the cytochrome c, as determined by spectral analysis at room temperature. The cytochrome b-containing precipitate is removed by centrifugation at 1000 g for 10 min. The peptide composition of these fractions in which complete separation of the cytochromes was achieved is shown in Figs. 1B and 1C. Step 2. Separation of Core Protein. 14 The supernatant material (400 /zl) from step 1 containing cytochrome c, (Fig. 1C) is treated with 120 ~1 of sodium taurocholate, 20/xl of 2 M Tris-chloride, pH 8.3, a few grains of dithionite to reduce the complex, and 10/xl of mersalyl solution. The mixture is incubated for 3 hr at 0o--4°, and the white precipitate containing core protein is removed by centrifugation at 1000 g for 10 min. The precipitate is washed twice with 0.8 ml of water before electrophoresis (Fig. 1D). z0j. S. Rieske, and W. S. Zaugg,Biochern. Biophys. Res. Commun. 8, 421 (1%2). 2, j. S. Rieske, H. Baum, C. D. Stoner, and S. H. Lipton,J. Biol. Chem. 242, 4854 (1967).
84
ELECTRON TRANSFER COMPLEXES
[13]
Step 3. Separation o f the Cytochrome cl Heme-Binding Peptide. 4"8 The supernatant obtained in step 2 after removal of core protein is further incubated for 48 hr at 00-4 °. A bright red precipitate is formed which is removed by centrifugation as above and is washed once in 400 /.d of water. This precipitate contains most of the cytochrome cl heme-binding peptide (Fig. 1E). If core protein is not completely removed from solution in step 2, small amounts of this peptide can subsequently appear with the cytochrome cl heme-binding peptide. This can be circumvented if the sample is again centrifuged a few hours after removal of core protein and the resulting pellet is discarded. The subsequent precipitate that forms contains little or no contaminating core protein. The supernatant obtained after removal of the heme-binding peptide is deficient in this peptide and is enriched with the small molecular weight peptides (Fig. 1F). Step 4. Separation of Crude Iron-Sulfur Protein. 2"6"18 To 100 p~l of complex III (16 mg of protein per milliliter) suspended in 0.66 M sucrose, 50 mM Tris-chloride, pH 8.0, and 1 mM histidine, are added 6 /~1 of antimycin solution, 10/~1 of taurocholate, and 27/~1 of saturated ammonium sulfate. The mixture is incubated for 30 min at 25 °, and the brownish precipitate containing iron-sulfur protein is removed by centrifugation at 1000 g for 10 min and washed once with 400 ~1 of water. The peptides of this fraction are shown in Fig. IG. Step 5. Separation of Cytochrome b. 22 Complex III (200/~1 of a 16 mg/ml protein solution) is removed from Tris-histidine-sucrose buffer by precipitation with 50% saturated ammonium sulfate, and is suspended in 0.1 M potassium phosphate buffer, pH 7.4. To this is added 48/~1 of potassium cholate (20%, w/v) and 37/~1 of saturated ammonium sulfate. The mixture is heated to 40 ° until a precipitate starts to form and is then placed at 20 ° for 5 min. The material which is removed by centrifugation at 60,000 g for 30 min is suspended in 100/~1 of 0.25 M sucrose and is dissolved by adding 3/.~1 of 10% (w/v) SDS. Solid ammonium sulfate (2.6 mg) is added and the solution is then held on ice for 10 min and centrifuged as above. The resulting pellet is again dissolved in 100/~1 of 0.25 M sucrose and 3 /_tl of 10% SDS and then frozen at - 2 0 °. The thawed solution is centrifuged as above and the resulting supernatant (100/~1) is treated with 2.6 mg of solid ammonium sulfate and centrifuged once more. The pellet obtained after centrifugation is suspended in 100/~! of 0.25 M sucrose, 3 p~l of SDS, and 2.6 mg of ammonium sulfate, and centrifuged a final time to give the cytochrome b pellet (Fig. 1H). 22 R. Goldberger, A. L. Smith, H. Tisdale, and R. Bomstein, J. Biol. Chem. 236, 2788 (1961).
[13]
COMPLEX II1 FROM BEEF HEART
85
Comments on the Separation of Peptides from Complex III Cytochrome b and cytochrome c ~can also be separated if taurocholate is replaced in the above scheme (step 1) with guanidine-HC1, za The rate of separation of the two cytochromes has been studied, 2~ and shown to be maximal at 10-12% taurocholate or 1 M guanidine-HCl and 25-30% saturation with ammonium sulfate. Binding of antimycin (stoichiometric v~ith cytochrome cl content) or reduction of the complex with dithionite prevent splitting of the cytochromes.21 The fractionation scheme presented above provides a rapid method by which certain peptides of complex III can be highly enriched, and in some cases purified. The chief advantages of the procedure are that of speed and the scale on which one can work. A few milligrams of complex III protein can be used for the complete fractionation, and the procedure can be scaled up as much as necessary. It provides the starting point for purification of the iron-sulfur protein, 18 and it yields high-purity preparations of cytochrome ct heme-binding peptide as well as core protein. The two core proteins 4"14 are not separated from each other by this technique. Larger scale gel filtration procedures in the presence of SDS and or guanidine-HC1 have also been reported for the partial separation of the peptides from Complex III. 5 Cytochrome Composition of Complex III Complex III contains 6-8 nmol of cytochrome b per milligram of protein and 3-4 nmol of cytochrome cl per milligram of protein as determined by their reduced-oxidized difference spectra at 562-575 nm and 554-540 nm, respectively. 23 At least two b-cytochromes can be resolved spectrally,23-25 potentiometrically,2~, 25 and on the basis of their reduction patterns with various reductants. 23'24 These characteristics are summarized in Table I. Resolution of Complex III: A Comparison of Methods Several preparations of complex III have been reported, the properties of which differ only in detail. 9' 22,26 The methods of preparation are 23 B. D. N e l s o n 24 K. A. Davis, (1973). 25 p. Riccio, H. 250 (1977). 2n y . Hatefi, A.
and P. Gellerfors, Biochim. Biophys. Acta 357, 358 (1974). Y. Hatefi, K. L. Poff, and W. L. Butler, Biochim. Biophys. Acta 325, 341 Sch~igger, W. D. Engel, and G. v c n Jagow, Biochim. Biophys. Acta 459, G. Haavik, and D. E. Griffiths, J. Biol. Chem. 237, 1681 (1962).
86
[13]
ELECTRON TRANSFER COMPLEXES TABLE 1 PROPERTIES OF THE CYTOCHROMES OF ISOLATED COMPLEX III"
Cytochrome
Concentration (nmol/mg protein)
Absorption maximum of the u-band (nm)
EmT.2 (mV)
Cytochrome b-562
3-4
562
+90
Cytochrome b-565 Cytochrome cl
3-4 3-4
565 553
-34 +230
Reduced by DQH2, Q2H2, ascorbate + TMPD or PMS, succinate (slowly), and dithionite Dithionite Ascorbate, dithiothreitol, and reductants listed above
" From B. D. Nelson and P. Gellerfors, Biochim. Biophys. Acta 357, 358 (1974).
similar in principle, insofar as the complex is always separated from the other respiratory chain components by dissolution with cholate (varying between 0.25 and 1.0 mg per milligram of protein) and is precipitated from the inner membrane with ammonium sulfate (varying between 35% and 50% saturation). The most well documented preparations of complex III are described below. Of particular importance is the starting material used. This can be influential in selecting a method for isolation of complex III if a specific, additional respiratory chain complex is a desired byproduct of the fractionation. M e t h o d o f H a t e f i et al. 26
The starting material for this preparation of complex III is N A D H cytochrome c reductase (fraction R4B) isolated by Hatefi et al. 27 To obtain R4B, however, it is necessary in the final steps of its preparation to precipitate it with 3.2 ml of 50% saturated ammonium acetate per 100 ml of supernatant 27 rather than 3.2 ml per milliliter of supernatant as described previously.2S To prepare complex III, the R4B pellet is diluted to 10 mg of protein per milliliter in 0.66 M sucrose, 50 mM Tris-chloride, pH 8.0, and 1 mM histidine (Tris-sucrose-histidine buffer). Neutral potassium cholate (20% w/v) is added to give 0.4 mg of cholate per milligram of protein, followed by saturated ammonium sulfate (0 °) to a final concentration of 6.5 ml per 100 ml of protein suspension. After 15 min at 0 °27 y . Hatefi, A. G. Haavik, and P. Jurtshuk, Biochim. Biophys. Acta 52, 106 (1961). 2, y . Hatefi and J. R. Rieske, this series, Vol. 10, p. 225.
[13]
COMPLEX III FROM BEEF HEART
87
4 ° this suspension is centrifuged at 41,000 g for 15 min to remove complex I, and the supernatant containing complex III is again treated with ammonium sulfate (6.9 ml per 100 ml protein suspension). This is centrifuged as before, and the resulting supernatant is treated with saturated ammonium sulfate (8 ml per 100 ml of supernatant). After a final centrifugation as described above, complex III collects near the surface as an oily material. M e t h o d o f R i e s k e et al. 2
This preparation of complex III uses the supernatant solution obtained after removal of fraction R4B (NADH-cytochrome c reductase ~-6) as its starting material. Thus, the same precautions regarding the concentrations of ammonium acetate used in the preparation of R4B (see above) must be heeded. To the supernatant obtained after removal of R4B is added 450 g of crystalline ammonium acetate per liter of supernatant. After standing 10 min the sample is centrifuged at 79,000 g for 40 min and the pellet is suspended in Tris-sucrose-histidine buffer and stored overnight at - 2 0 °. The thawed pellet is diluted to 10 mg of protein per milliliter, and potassium cholate (20% w/v) is added to give 0.25 mg per milligram of protein, followed by neutral ammonium sulfate to give 35% saturation (54 ml per 100 ml of protein solution). This suspension is centrifuged for 15 min at 79,000 g. The resulting supernatant solution is taken to 40% saturation with ammonium sulfate (8.35 ml per 100 ml of protein solution) and centrifuged as before. The floating, oily material is removed by filtration through glass wool. The remaining supernatant is taken to 42% saturation with ammonium sulfate (3.45 ml per 100 ml of protein solution) and centrifuged as in the previous step. The floating, oily material is discarded, and the supernatant is taken to 48% saturation with ammonium sulfate (11.5 ml per 100 ml of protein solution). The resulting precipitate, which contains complex III, is suspended in Trissucrose-histidine buffer (approximately 20 mg of protein per milliliter) and stored at - 2 0 °. If any, or all, of the complex III is found floating after the final precipitation step, this can be recovered by filtration through glass wool followed by extraction of the glass wool with 3 × 0.5 ml of Tris-sucrose-histidine buffer. M e t h o d ~1" Yu et al. 9
The starting material for this preparation is succinate-cytochrome c reductase, which is solubilized from beef heart muscle preparation with 1% cholate (0.5 mg per milligram of protein) and, after several additional steps, precipitated between 35% and 50% saturation with neutralized ammonium sulfate. 9 Succinate-cytochrome c reductase thus obtained is
88
ELECTRON TRANSFER COMPLEXES
[13]
diluted to 5 mg of protein per milliliter in 50 mM phosphate buffer, pH 7.4. This suspension is incubated for 30 min with 20 mM succinate, after which the pH is adjusted to 10.5 with 1 N NaOH and incubation is continued for an additional 30 min under anaerobiasis. The sample is then centrifuged at 200,000 g for 90 min to remove solubilized succinate dehydrogenase. The pellet is suspended by homogenization in deoxygenated 50 mM phosphate buffer, pH 7.4, adjusted to pH 10.5 and immediately centrifuged as above. The resulting pellet is washed once with phosphate buffer, pH 7.4, and then diluted to 10 mg of protein per milliliter in 0.9% KCI. Potassium cholate (10%, pH 9.0) is added to give 0.3 mg per milligram of protein and the sample is centrifuged. The resulting supernatant is brought to 50% saturation with saturated, neutralized ammonium sulfate. The pellet obtained by centrifugation is suspended in 25 mM potassium phosphate buffer, pH 7.4, containing 0.25 M sucrose. The insoluble material is removed by centrifugation, and the red supernatant is designated soluble cytochrome bcl complex. M e t h o d o f Riccio et al. 2~,29
This method differs in principle from those listed above and with modification should be helpful in isolating complex III on a small scale. Submitochondrial particles treated with antimycin are solubilized in 4.8% Triton X-100 (1.37 mg per milligram of protein) and 0.5 M NaC1. The solubilized proteins are placed on a hydroxyapatite column (4 mg of protein per milliliter of gel) containing 110 mM sodium phosphate buffer, 90 mM NaCI, 1 mM EDTA, and 0.5% Triton X-100, pH 7.4, and are eluted with the same solution. Elution is stopped after the bulk of the cytochrome oxidase is removed. At this point about 70% of the total protein has been removed from the column. The column buffer is exchanged with buffer containing 10 mM sodium phosphate, 1 mM EDTA, and 0.2% Triton X-100 in order to decrease the salt and detergent concentrations of the column. Complex III is now specifically eluted using the latter buffer containing 8 mM citrate. Comments on the Various Preparations o f Complex III
Table II summarizes the properties of different preparations of complex III. It should be noted that some variation in these values can be expected within each individual preparation of complex III, regardless of the methods of isolation chosen. The following general conclusions can be drawn. 2~ G. von Jagow, H. Schfigger, P. Riccio, M. Klingenberg, and H. J. Kolb, Biochim. Biophys. Acta 462, 549 (1977).
[13]
COMPLEX
111 F R O M
BEEF
HEART
89
0
~2
>, m ,1 ,..4,
0
~
I~-
("4
0 q
0 •-
~,~ ~
'
.
e~ c~
~ ~
~
0 b"
ul
"
#
© Z
~
Z
.~
=[.,-
~
:>-. '.-,
0 r,.)
7 ~
s N
•
E ~~ ~~ ~ ~ " ~o ~E .~ ~ E ~' ~ ~~, E'-.
•
,
EEE=~,-.
0
I=
0
90
ELECTRON TRANSFER COMPLEXES
[13]
The QH2-cytochrome c reductase activities of the Hatefi et al. 26 and Rieske et al. 2 preparations are similar, and both appear to be higher than that in the preparation of Yu et al.9 The Rieske preparation contains more flavin and is slightly more contaminated with succinate dehydrogenase than the preparations either of Hatefi or of Yu. The polypeptide composition of all three preparations are similar. '~The yield of the Rieske preparation is higher than that of the Hatefi preparation2; yields of the preparation of Yu et al.9 were not given. The main advantage of the preparation of Yu et al.9 appears to be its ability to recombine with purified succinate dehydrogenase to form succinate-cytochrome c reductase. In general, there seem to be more similarities than differences among the three above-mentioned preparations. In contrast, the preparation of Riccio et al. 25 differs in several important respects. The first is that it contains antimycin and is therefore enzymically inactive. It also lacks two polypeptides. However, the method provides a new and important approach for isolating complex III. In addition, small amounts of starting material can be used with this method, and the yields of the complex are high. The complex as isolated 25 is highly purified with respect to the cytochromes and is free from contaminating peptides. It should provide an excellent starting point for preparation of cytochromes b and cl.
Assay Complex III (QH2-cytochrome c reductase) was isolated on the basis of its specificity toward short-chained ubiquinone homologs. 2"26 Since the preferred substrates (QI-Q~) are not commercially available, several investigators have turned to duroquinol as an alternative substrate. Duroquinol is, however, easily air-oxidized and requires special handling. Preparation o f Duroquinol
Duroquinone (tetramethyl-p-benzoquinone) is available commercially and can be used without further purification. Sufficient duroquinone is dissolved in 5-10 ml of methanol to give a final concentration of 30 mM. Reduction is achieved with a few grains of borohydride under constant stirring and under a stream of nitrogen. Complete reduction is assumed when the faint yellow disappears. The pH, which is continuously monitored with a pH electrode, is between 10 and 11, and is now lowered to pH 2-3 with 10-20/xl of 3 N HC1. At this point excess borohydride is eliminated. This solution of duroquinol, when tightly capped and placed in an ice bath, remains reduced for several hours. If duroquinol is to be
[13]
COMPLEX Ill FROM BEEF HEART
91
used to drive the redox-driven proton pump in complex III vesicles, 3°' 31 the pH should be adjusted to pH 6 or 6.5 with a few microliters of 1 N NaOH; otherwise it may be used as prepared above. Fresh duroquinol should be prepared every 5-6 hr, or as soon as the pale yellow starts to return to the solution. Procedure
All measurements are made at 550 minus 540 nm with the full scale of the recorder adjusted to 0.1 or 0 . 2 0 D units. To 1 ml of assay medium containing 25 mM potassium phosphate buffer, pH 7.4, 50/zM EDTA, and 14/xM cytochrome c (0.2 mg/ml) is added 2/xl of duroquinol (60/zM final concentration) from a microsyringe. The nonenzymic reduction of cytochrome c is recorded. Complex III (5-20/xg of protein) is immediately added to initiate enzymic reduction of cytochrome c. The enzymic rates must always be corrected for the nonenzymic reduction of cytochrome c. The latter is sensitive to concentrations of duroquinol and cytochrome c and to pH. An increase in any of these can result in such a high nonenzymic rate as to mask the reaction catalyzed by complex Ill. The conditions given above allow determination of the initial, linear, portion of the enzymic reaction. Comments
Duroquinol-cytochrome c reductase activity varies from 1 to 8/xmol of cytochrome c reduced per minute per milligram of protein in different preparations of complex III when assayed at fixed concentrations of duroquinol. Rates obtained with duroquinol are much slower than those obtained with Q2H2 (Vmax = 200--1000/xmol/min per milligram of protein), and duroquinol is therefore not the substrate of choice for assaying complex III if short-chain ubiquinone homologs are available. Duroquinol can be used as effectively, however, to drive the redox-driven proton pump and to demonstrate "respiratory control" in phospholipid vesicles reconstituted with complex III. 3°'31 Duroquinol-cytochrome c reductase activity is completely inhibited by antimycin.
a0 K. H. L e u n g and P. C. Hinkle, J. Biol. Chem. 250, 8467 (1975). :31 F. Guerrieri and B. D. N e l s o n , FEBS Lett. 54, 339 (1975).
[13]
ELECTRON TRANSFER COMPLEXES
[13] C h a r a c t e r i z a t i o n and Resolution of Complex from Beef Heart Mitochondria
By
III
B . D E A N NELSON a n d P,~R GELLERFORS
Peptide Composition of Complex III Peptide Composition of Complex III as Determined by Sodium Dodecyl Sulfate(SDS)-Gel Electrophoresis
Electrophoretic Procedures Complex III is dissolved in a mixture of 2% SDS, 5% 2-mercaptoethanol, and 10 mM Tris-chloride, pH 6.8, to a final concentration of 1-2 mg of protein per milliliter. The solution is incubated for 3 rain at 100°, and 20-50 tzg of protein are applied to 12.5% (w/v) polyacrylamide gels containing a constant ratio of acrylamide to methylene bisacrylamide of 37 : 1. Preparation of the gels and electrophoresis are carried out according to the methods of Laemmli.l
Major Peptides of Complex Ili Complex III z contains seven major peptides (Fig. 1A), the molecular weights of which are approximately 50,000, 47,000, 29,000, 26,000, 15,000, 13,000, and 10,000 when separated by electrophoresis as described above. The molecular weight of 10,000 for the last peptide appears to be overestimated in this system. In more highly cross-linked gels, this band can be resolved into several peptides with molecular weights less than 10,000. a.5 The identities of the above peptides are far from certain, and in very few cases can a function be absolutely associated with each fraction. Furthermore, rigorous analysis showing peptide homogeneity within each band has not been accomplished. On the other hand, all complex III preparations so far reported from beef heart 3-11 or yeast TM mitochondria i U. K. Laemmli, Nature (London) 227, 680 (1970). 2 j. S. Rieske, W. S. Zaugg, and R. E. Hansen, J. Biol. Chem. 239, 3023 (1964). :t C. A. M. Marres and E. C. Slater, Biochim. Biophys. Acta 462, 531 (1977). 4 p. Gellerfors and B. D. Nelson, Eur. J. Biochem. 52, 433 (1975). 5 R. L. Bell and R. A. Capaldi, Biochemistry 15, 996 (1976). 6 H. Baum, H. I. Silman, J. S. Rieske, and S. H. Lipton, J. Biol. Chem. 242, 4876 (1967). 7 U. D. Gupta, and J. S. Rieske, Biochem. Biophys. Res. Commun. 54, 1247 (1973). 8 j. F. Hare and F. L. Crane, Sub-Cell. Biochem. 3, 1 (1974).
[13]
81
COMPLEX 11t FROM BEEF HEART
A
B
C
D
E
F
G
H
50,000 47,000 29,000 26,000
"
W
o ~!iiii~/
~
i~ii!i~I
15,000 13,000 10,000
FIG. I. Separation of the peptides of complex 111. Separation of the peptides and sodium dodecyl sulfate (SDS)-gel electrophoresis were carried out as described in the text. A, Complex lIl: B, cytochrome b fraction after splitting the complex; C, cytochrome cl fraction after splitting the complex; D, core protein; E, cytochrome cl heme-binding peptide; F, supernatant after removal of cytochrome c, heine-binding peptide: G, iron-sulfur protein; H, cytochrome b fraction.
have a characteristic peptide pattern similar to that shown in Fig. 1A with respect to the relative positions of the peptides on the gels and to their relative staining intensities. The major peptides listed above probably represent the basic peptide unit of complex III. With the above reservations, the major peptides can be given an assignment based upon the enrichment or isolation of their prosthetic groups. The molecular weight (M0 estimates differ slightly in various laboratories,3-~, 7-11,13 but the peptides can be identified by their charac9 C. A. Yu, L. Yu, and T. E. King, J. Biol. Chem. 249, 4905 (1974). lo R. A. Capaldi, Arch. Biochem. Biophys. 163, 99 (1974). u E. C. Slater, in "Electron Transfer Chains and Oxidative Phosphorylation" (E. Quagliariello, s. Papa, F. Palmieri, E. C. Slater, and N. Siliprandi, eds.), p. 3. North-Holland Publ., Amsterdam, 1975. 12 M. B. Katan, L. Pool, and G. S. P. Groot, Eur. J. Biochem. 65, 95 (1976). 13 R. A. Capaldi, R. L. Bell, and T. Branchek, Biochem. Biophys. Res. Commun. 74, 425 (1977).
82
ELECTRON TRANSFER COMPLEXES
[13]
teristic distributions on the gels and their staining intensities. Core proteins 1 and 2 (Mr 50,000 and 47,000) contain no prosthetic groups 14 but appear to be normal constituents of complex III, since their removal leads to loss of antimycin binding, ~ and since specific alkylation of core protein-1 with iodoacetamide leads to inactivation of enzyme activity.lG The Mr 29,000 peptide contains cytochrome ¢1 heme-binding peptide, which, because of the covalently bound heme, can be located directly on the gels. It has been suggested that this fraction, in analogy with Neurospora,~7 also contains cytochrome b. 3 The Mr 26,000 peptide corresponds to the isolated iron-sulfur protein. 2-4' s-10,18 The lower part of the gel contains 3 major peptides whose functions are not known and the identities of which have not been completely established. The smallest of these (Mr 10,000) has been associated with the binding of antimycin. 7 This band contains, however, several polypeptides with molecular weights of less than 10,000. 3"~ The remaining peptides (Mr 15,000 and 13,000) have been associated with cytochromes b or cl. Some confusion has arisen regarding these latter peptides, since they can switch positions on a gel, depending upon the electrophoretic system used.13 Additional peptides can also appear on the gels, especially in the area above Mr 60,000. They never make up more than 2% of the stainable protein and are often missing from the gels. They probably represent contaminations. Among the most prominent of these is a peptide of Mr 70,000, which is either the large subunit of succinate dehydrogenase 19 or an artifact of the electrophoretic system. 4 Peptides previously reported 4 in the molecular weight ranges of 17,000-18,000 and 34,000-37,000 are seldom observed as long as the samples are prepared for electrophoresis and the electrophoresis is carried out according to the procedures outlined above. The peptide pattern on SDS-gel is altered only slightly when the samples are prepared and electrophoresed in the absence of mercaptoethanol, indicating the absence of intermolecular disulfide bands. SDSgels stained for carbohydrate using periodic acid-Schiff reagent (PAS) show no positive staining reactions associated with the peptide bands. A positive reaction is observed near the dye front and probably represents carbohydrate-containing lipids. Molar ratios of the peptides have been estimated 3"4 to be: 50,000 (1 14 H. I. Silman, J. S. Rieske, S. H. Lipton, and H. B a u m , J. Biol. Chem. 242, 4867 (1967). 15 j. S. Rieske, S. H. Lipton, H. B a u m , and H. I. Silman, J. Biol. Chem. 242, 4888 (1967). 16 p. Gellerfors, M. L u n d 6 n , and B. D. Nelson, Eur. J. Biochem. 67, 463 (1976). lr H. Weiss, Biochim. Biophys. Acta 456, 291 (1976). 18 j. S. Rieske, D. H. M a c L e n n a n , and R. Colman, Biochem. Biophys. Res. Commun. 15, 338 (1964). 19 K. A. Davis, and Y. Hatefi, Biochemistry 10, 2509 (1971).
[13]
COMPLEXill FROMBEEF HEART
83
mol), 47,000 (1 mol), 29,000 (1 tool), 26,000 (1-2 mol), 15,000 (1 mol), 13,000 (2 mol), and 10,000 (2-6 tool). Isolation of the Peptides of Complex III Reagents 1. Sodium taurocholate (DIFCO), a 30% (w/v)solution in water 2. Ammonium sulfate, saturated at 00-4 ° and neutralized with ammonia 3. Imidazole, 1 M solution buffered to pH 6.6 with sulfuric acid 4. Antimycin, 1 mg/ml in ethanol 5. Sodium mersalyl, 50 mM solution in water 6. Sodium dodecyl sulfate (SDS), 10% (w/v) in water 7. Potassium cholate, recrystallized in 70% ethanol, 20% (w/v) in water Isolation Scheme Step 1. Separation of the Cytochrome b and Cytochrome c, fractions. 2°'21 To 100/xl of complex III (3 mg of protein) suspended in 0.66 M sucrose and 50 mM Tris-chloride, pH 8.0, is added 10/xl of imidazolesulfate solution followed by 110 tzl of water and 120 /zl of saturated ammonium sulfate. Separation of the cytochromes is initiated by addition of 60 p,1 of the taurocholate solution. After addition of taurocholate, the mixture is taken to 25 ° and incubated for 3 hr. The precipitate which forms contains 80-100% of the cytochrome b, and the supernatant contains 80-100% of the cytochrome c, as determined by spectral analysis at room temperature. The cytochrome b-containing precipitate is removed by centrifugation at 1000 g for 10 min. The peptide composition of these fractions in which complete separation of the cytochromes was achieved is shown in Figs. 1B and 1C. Step 2. Separation of Core Protein. 14 The supernatant material (400 /zl) from step 1 containing cytochrome c, (Fig. 1C) is treated with 120 ~1 of sodium taurocholate, 20/xl of 2 M Tris-chloride, pH 8.3, a few grains of dithionite to reduce the complex, and 10/xl of mersalyl solution. The mixture is incubated for 3 hr at 0o--4°, and the white precipitate containing core protein is removed by centrifugation at 1000 g for 10 min. The precipitate is washed twice with 0.8 ml of water before electrophoresis (Fig. 1D). z0j. S. Rieske, and W. S. Zaugg,Biochern. Biophys. Res. Commun. 8, 421 (1%2). 2, j. S. Rieske, H. Baum, C. D. Stoner, and S. H. Lipton,J. Biol. Chem. 242, 4854 (1967).
84
ELECTRON TRANSFER COMPLEXES
[13]
Step 3. Separation o f the Cytochrome cl Heme-Binding Peptide. 4"8 The supernatant obtained in step 2 after removal of core protein is further incubated for 48 hr at 00-4 °. A bright red precipitate is formed which is removed by centrifugation as above and is washed once in 400 /.d of water. This precipitate contains most of the cytochrome cl heme-binding peptide (Fig. 1E). If core protein is not completely removed from solution in step 2, small amounts of this peptide can subsequently appear with the cytochrome cl heme-binding peptide. This can be circumvented if the sample is again centrifuged a few hours after removal of core protein and the resulting pellet is discarded. The subsequent precipitate that forms contains little or no contaminating core protein. The supernatant obtained after removal of the heme-binding peptide is deficient in this peptide and is enriched with the small molecular weight peptides (Fig. 1F). Step 4. Separation of Crude Iron-Sulfur Protein. 2"6"18 To 100 p~l of complex III (16 mg of protein per milliliter) suspended in 0.66 M sucrose, 50 mM Tris-chloride, pH 8.0, and 1 mM histidine, are added 6 /~1 of antimycin solution, 10/~1 of taurocholate, and 27/~1 of saturated ammonium sulfate. The mixture is incubated for 30 min at 25 °, and the brownish precipitate containing iron-sulfur protein is removed by centrifugation at 1000 g for 10 min and washed once with 400 ~1 of water. The peptides of this fraction are shown in Fig. IG. Step 5. Separation of Cytochrome b. 22 Complex III (200/~1 of a 16 mg/ml protein solution) is removed from Tris-histidine-sucrose buffer by precipitation with 50% saturated ammonium sulfate, and is suspended in 0.1 M potassium phosphate buffer, pH 7.4. To this is added 48/~1 of potassium cholate (20%, w/v) and 37/~1 of saturated ammonium sulfate. The mixture is heated to 40 ° until a precipitate starts to form and is then placed at 20 ° for 5 min. The material which is removed by centrifugation at 60,000 g for 30 min is suspended in 100/~1 of 0.25 M sucrose and is dissolved by adding 3/.~1 of 10% (w/v) SDS. Solid ammonium sulfate (2.6 mg) is added and the solution is then held on ice for 10 min and centrifuged as above. The resulting pellet is again dissolved in 100/~1 of 0.25 M sucrose and 3 /_tl of 10% SDS and then frozen at - 2 0 °. The thawed solution is centrifuged as above and the resulting supernatant (100/~1) is treated with 2.6 mg of solid ammonium sulfate and centrifuged once more. The pellet obtained after centrifugation is suspended in 100/~! of 0.25 M sucrose, 3 p~l of SDS, and 2.6 mg of ammonium sulfate, and centrifuged a final time to give the cytochrome b pellet (Fig. 1H). 22 R. Goldberger, A. L. Smith, H. Tisdale, and R. Bomstein, J. Biol. Chem. 236, 2788 (1961).
[13]
COMPLEX II1 FROM BEEF HEART
85
Comments on the Separation of Peptides from Complex III Cytochrome b and cytochrome c ~can also be separated if taurocholate is replaced in the above scheme (step 1) with guanidine-HC1, za The rate of separation of the two cytochromes has been studied, 2~ and shown to be maximal at 10-12% taurocholate or 1 M guanidine-HCl and 25-30% saturation with ammonium sulfate. Binding of antimycin (stoichiometric v~ith cytochrome cl content) or reduction of the complex with dithionite prevent splitting of the cytochromes.21 The fractionation scheme presented above provides a rapid method by which certain peptides of complex III can be highly enriched, and in some cases purified. The chief advantages of the procedure are that of speed and the scale on which one can work. A few milligrams of complex III protein can be used for the complete fractionation, and the procedure can be scaled up as much as necessary. It provides the starting point for purification of the iron-sulfur protein, 18 and it yields high-purity preparations of cytochrome ct heme-binding peptide as well as core protein. The two core proteins 4"14 are not separated from each other by this technique. Larger scale gel filtration procedures in the presence of SDS and or guanidine-HC1 have also been reported for the partial separation of the peptides from Complex III. 5 Cytochrome Composition of Complex III Complex III contains 6-8 nmol of cytochrome b per milligram of protein and 3-4 nmol of cytochrome cl per milligram of protein as determined by their reduced-oxidized difference spectra at 562-575 nm and 554-540 nm, respectively. 23 At least two b-cytochromes can be resolved spectrally,23-25 potentiometrically,2~, 25 and on the basis of their reduction patterns with various reductants. 23'24 These characteristics are summarized in Table I. Resolution of Complex III: A Comparison of Methods Several preparations of complex III have been reported, the properties of which differ only in detail. 9' 22,26 The methods of preparation are 23 B. D. N e l s o n 24 K. A. Davis, (1973). 25 p. Riccio, H. 250 (1977). 2n y . Hatefi, A.
and P. Gellerfors, Biochim. Biophys. Acta 357, 358 (1974). Y. Hatefi, K. L. Poff, and W. L. Butler, Biochim. Biophys. Acta 325, 341 Sch~igger, W. D. Engel, and G. v c n Jagow, Biochim. Biophys. Acta 459, G. Haavik, and D. E. Griffiths, J. Biol. Chem. 237, 1681 (1962).
86
[13]
ELECTRON TRANSFER COMPLEXES TABLE 1 PROPERTIES OF THE CYTOCHROMES OF ISOLATED COMPLEX III"
Cytochrome
Concentration (nmol/mg protein)
Absorption maximum of the u-band (nm)
EmT.2 (mV)
Cytochrome b-562
3-4
562
+90
Cytochrome b-565 Cytochrome cl
3-4 3-4
565 553
-34 +230
Reduced by DQH2, Q2H2, ascorbate + TMPD or PMS, succinate (slowly), and dithionite Dithionite Ascorbate, dithiothreitol, and reductants listed above
" From B. D. Nelson and P. Gellerfors, Biochim. Biophys. Acta 357, 358 (1974).
similar in principle, insofar as the complex is always separated from the other respiratory chain components by dissolution with cholate (varying between 0.25 and 1.0 mg per milligram of protein) and is precipitated from the inner membrane with ammonium sulfate (varying between 35% and 50% saturation). The most well documented preparations of complex III are described below. Of particular importance is the starting material used. This can be influential in selecting a method for isolation of complex III if a specific, additional respiratory chain complex is a desired byproduct of the fractionation. M e t h o d o f H a t e f i et al. 26
The starting material for this preparation of complex III is N A D H cytochrome c reductase (fraction R4B) isolated by Hatefi et al. 27 To obtain R4B, however, it is necessary in the final steps of its preparation to precipitate it with 3.2 ml of 50% saturated ammonium acetate per 100 ml of supernatant 27 rather than 3.2 ml per milliliter of supernatant as described previously.2S To prepare complex III, the R4B pellet is diluted to 10 mg of protein per milliliter in 0.66 M sucrose, 50 mM Tris-chloride, pH 8.0, and 1 mM histidine (Tris-sucrose-histidine buffer). Neutral potassium cholate (20% w/v) is added to give 0.4 mg of cholate per milligram of protein, followed by saturated ammonium sulfate (0 °) to a final concentration of 6.5 ml per 100 ml of protein suspension. After 15 min at 0 °27 y . Hatefi, A. G. Haavik, and P. Jurtshuk, Biochim. Biophys. Acta 52, 106 (1961). 2, y . Hatefi and J. R. Rieske, this series, Vol. 10, p. 225.
[13]
COMPLEX III FROM BEEF HEART
87
4 ° this suspension is centrifuged at 41,000 g for 15 min to remove complex I, and the supernatant containing complex III is again treated with ammonium sulfate (6.9 ml per 100 ml protein suspension). This is centrifuged as before, and the resulting supernatant is treated with saturated ammonium sulfate (8 ml per 100 ml of supernatant). After a final centrifugation as described above, complex III collects near the surface as an oily material. M e t h o d o f R i e s k e et al. 2
This preparation of complex III uses the supernatant solution obtained after removal of fraction R4B (NADH-cytochrome c reductase ~-6) as its starting material. Thus, the same precautions regarding the concentrations of ammonium acetate used in the preparation of R4B (see above) must be heeded. To the supernatant obtained after removal of R4B is added 450 g of crystalline ammonium acetate per liter of supernatant. After standing 10 min the sample is centrifuged at 79,000 g for 40 min and the pellet is suspended in Tris-sucrose-histidine buffer and stored overnight at - 2 0 °. The thawed pellet is diluted to 10 mg of protein per milliliter, and potassium cholate (20% w/v) is added to give 0.25 mg per milligram of protein, followed by neutral ammonium sulfate to give 35% saturation (54 ml per 100 ml of protein solution). This suspension is centrifuged for 15 min at 79,000 g. The resulting supernatant solution is taken to 40% saturation with ammonium sulfate (8.35 ml per 100 ml of protein solution) and centrifuged as before. The floating, oily material is removed by filtration through glass wool. The remaining supernatant is taken to 42% saturation with ammonium sulfate (3.45 ml per 100 ml of protein solution) and centrifuged as in the previous step. The floating, oily material is discarded, and the supernatant is taken to 48% saturation with ammonium sulfate (11.5 ml per 100 ml of protein solution). The resulting precipitate, which contains complex III, is suspended in Trissucrose-histidine buffer (approximately 20 mg of protein per milliliter) and stored at - 2 0 °. If any, or all, of the complex III is found floating after the final precipitation step, this can be recovered by filtration through glass wool followed by extraction of the glass wool with 3 × 0.5 ml of Tris-sucrose-histidine buffer. M e t h o d ~1" Yu et al. 9
The starting material for this preparation is succinate-cytochrome c reductase, which is solubilized from beef heart muscle preparation with 1% cholate (0.5 mg per milligram of protein) and, after several additional steps, precipitated between 35% and 50% saturation with neutralized ammonium sulfate. 9 Succinate-cytochrome c reductase thus obtained is
88
ELECTRON TRANSFER COMPLEXES
[13]
diluted to 5 mg of protein per milliliter in 50 mM phosphate buffer, pH 7.4. This suspension is incubated for 30 min with 20 mM succinate, after which the pH is adjusted to 10.5 with 1 N NaOH and incubation is continued for an additional 30 min under anaerobiasis. The sample is then centrifuged at 200,000 g for 90 min to remove solubilized succinate dehydrogenase. The pellet is suspended by homogenization in deoxygenated 50 mM phosphate buffer, pH 7.4, adjusted to pH 10.5 and immediately centrifuged as above. The resulting pellet is washed once with phosphate buffer, pH 7.4, and then diluted to 10 mg of protein per milliliter in 0.9% KCI. Potassium cholate (10%, pH 9.0) is added to give 0.3 mg per milligram of protein and the sample is centrifuged. The resulting supernatant is brought to 50% saturation with saturated, neutralized ammonium sulfate. The pellet obtained by centrifugation is suspended in 25 mM potassium phosphate buffer, pH 7.4, containing 0.25 M sucrose. The insoluble material is removed by centrifugation, and the red supernatant is designated soluble cytochrome bcl complex. M e t h o d o f Riccio et al. 2~,29
This method differs in principle from those listed above and with modification should be helpful in isolating complex III on a small scale. Submitochondrial particles treated with antimycin are solubilized in 4.8% Triton X-100 (1.37 mg per milligram of protein) and 0.5 M NaC1. The solubilized proteins are placed on a hydroxyapatite column (4 mg of protein per milliliter of gel) containing 110 mM sodium phosphate buffer, 90 mM NaCI, 1 mM EDTA, and 0.5% Triton X-100, pH 7.4, and are eluted with the same solution. Elution is stopped after the bulk of the cytochrome oxidase is removed. At this point about 70% of the total protein has been removed from the column. The column buffer is exchanged with buffer containing 10 mM sodium phosphate, 1 mM EDTA, and 0.2% Triton X-100 in order to decrease the salt and detergent concentrations of the column. Complex III is now specifically eluted using the latter buffer containing 8 mM citrate. Comments on the Various Preparations o f Complex III
Table II summarizes the properties of different preparations of complex III. It should be noted that some variation in these values can be expected within each individual preparation of complex III, regardless of the methods of isolation chosen. The following general conclusions can be drawn. 2~ G. von Jagow, H. Schfigger, P. Riccio, M. Klingenberg, and H. J. Kolb, Biochim. Biophys. Acta 462, 549 (1977).
[13]
COMPLEX
111 F R O M
BEEF
HEART
89
0
~2
>, m ,1 ,..4,
0
~
I~-
("4
0 q
0 •-
~,~ ~
'
.
e~ c~
~ ~
~
0 b"
ul
"
#
© Z
~
Z
.~
=[.,-
~
:>-. '.-,
0 r,.)
7 ~
s N
•
E ~~ ~~ ~ ~ " ~o ~E .~ ~ E ~' ~ ~~, E'-.
•
,
EEE=~,-.
0
I=
0
90
ELECTRON TRANSFER COMPLEXES
[13]
The QH2-cytochrome c reductase activities of the Hatefi et al. 26 and Rieske et al. 2 preparations are similar, and both appear to be higher than that in the preparation of Yu et al.9 The Rieske preparation contains more flavin and is slightly more contaminated with succinate dehydrogenase than the preparations either of Hatefi or of Yu. The polypeptide composition of all three preparations are similar. '~The yield of the Rieske preparation is higher than that of the Hatefi preparation2; yields of the preparation of Yu et al.9 were not given. The main advantage of the preparation of Yu et al.9 appears to be its ability to recombine with purified succinate dehydrogenase to form succinate-cytochrome c reductase. In general, there seem to be more similarities than differences among the three above-mentioned preparations. In contrast, the preparation of Riccio et al. 25 differs in several important respects. The first is that it contains antimycin and is therefore enzymically inactive. It also lacks two polypeptides. However, the method provides a new and important approach for isolating complex III. In addition, small amounts of starting material can be used with this method, and the yields of the complex are high. The complex as isolated 25 is highly purified with respect to the cytochromes and is free from contaminating peptides. It should provide an excellent starting point for preparation of cytochromes b and cl.
Assay Complex III (QH2-cytochrome c reductase) was isolated on the basis of its specificity toward short-chained ubiquinone homologs. 2"26 Since the preferred substrates (QI-Q~) are not commercially available, several investigators have turned to duroquinol as an alternative substrate. Duroquinol is, however, easily air-oxidized and requires special handling. Preparation o f Duroquinol
Duroquinone (tetramethyl-p-benzoquinone) is available commercially and can be used without further purification. Sufficient duroquinone is dissolved in 5-10 ml of methanol to give a final concentration of 30 mM. Reduction is achieved with a few grains of borohydride under constant stirring and under a stream of nitrogen. Complete reduction is assumed when the faint yellow disappears. The pH, which is continuously monitored with a pH electrode, is between 10 and 11, and is now lowered to pH 2-3 with 10-20/xl of 3 N HC1. At this point excess borohydride is eliminated. This solution of duroquinol, when tightly capped and placed in an ice bath, remains reduced for several hours. If duroquinol is to be
[13]
COMPLEX Ill FROM BEEF HEART
91
used to drive the redox-driven proton pump in complex III vesicles, 3°' 31 the pH should be adjusted to pH 6 or 6.5 with a few microliters of 1 N NaOH; otherwise it may be used as prepared above. Fresh duroquinol should be prepared every 5-6 hr, or as soon as the pale yellow starts to return to the solution. Procedure
All measurements are made at 550 minus 540 nm with the full scale of the recorder adjusted to 0.1 or 0 . 2 0 D units. To 1 ml of assay medium containing 25 mM potassium phosphate buffer, pH 7.4, 50/zM EDTA, and 14/xM cytochrome c (0.2 mg/ml) is added 2/xl of duroquinol (60/zM final concentration) from a microsyringe. The nonenzymic reduction of cytochrome c is recorded. Complex III (5-20/xg of protein) is immediately added to initiate enzymic reduction of cytochrome c. The enzymic rates must always be corrected for the nonenzymic reduction of cytochrome c. The latter is sensitive to concentrations of duroquinol and cytochrome c and to pH. An increase in any of these can result in such a high nonenzymic rate as to mask the reaction catalyzed by complex Ill. The conditions given above allow determination of the initial, linear, portion of the enzymic reaction. Comments
Duroquinol-cytochrome c reductase activity varies from 1 to 8/xmol of cytochrome c reduced per minute per milligram of protein in different preparations of complex III when assayed at fixed concentrations of duroquinol. Rates obtained with duroquinol are much slower than those obtained with Q2H2 (Vmax = 200--1000/xmol/min per milligram of protein), and duroquinol is therefore not the substrate of choice for assaying complex III if short-chain ubiquinone homologs are available. Duroquinol can be used as effectively, however, to drive the redox-driven proton pump and to demonstrate "respiratory control" in phospholipid vesicles reconstituted with complex III. 3°'31 Duroquinol-cytochrome c reductase activity is completely inhibited by antimycin.
a0 K. H. L e u n g and P. C. Hinkle, J. Biol. Chem. 250, 8467 (1975). :31 F. Guerrieri and B. D. N e l s o n , FEBS Lett. 54, 339 (1975).