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Anti-cooperative Oxidation of Ubiquinol by the Yeast Cytochrome bc1 Complex

2004; Elsevier BV; Volume: 279; Issue: 15 Linguagem: Inglês

10.1074/jbc.m400193200

1083-351X

Raúl Covián, Emma Berta Gutiérrez-Cirlos, Bernard L. Trumpower,

Photosynthetic Processes and Mechanisms

We have investigated the interaction between monomers of the dimeric yeast cytochrome bc1 complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in cytochrome c1 reduction. At pH 8.8, where the redox potential of the iron-sulfur protein is ∼200 mV and in a bc1 complex with a mutated iron-sulfur protein of equally low redox potential, the amount of cytochrome c1 reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bc1 complex. Similar experiments in the presence of several equivalents of cytochrome c also showed only half of the bc1 complex participating in quinol oxidation. The extent of cytochrome b reduced corresponded to two bH hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two cytochrome b subunits. Antimycin stimulated the ubiquinol-cytochrome c reductase activity of the bc1 complex at low inhibitor/enzyme ratios. This stimulation could only be fitted to a model in which half of the bc1 dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the cytochrome b subunits of the dimer. These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bc1 complex dimer. We have investigated the interaction between monomers of the dimeric yeast cytochrome bc1 complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in cytochrome c1 reduction. At pH 8.8, where the redox potential of the iron-sulfur protein is ∼200 mV and in a bc1 complex with a mutated iron-sulfur protein of equally low redox potential, the amount of cytochrome c1 reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bc1 complex. Similar experiments in the presence of several equivalents of cytochrome c also showed only half of the bc1 complex participating in quinol oxidation. The extent of cytochrome b reduced corresponded to two bH hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two cytochrome b subunits. Antimycin stimulated the ubiquinol-cytochrome c reductase activity of the bc1 complex at low inhibitor/enzyme ratios. This stimulation could only be fitted to a model in which half of the bc1 dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the cytochrome b subunits of the dimer. These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bc1 complex dimer. The cytochrome bc1 complex transfers electrons from ubiquinol to cytochrome c by the protonmotive Q cycle mechanism (1Trumpower B.L. Gennis R.B. Annu. Rev. Biochem. 1994; 63: 675-716Crossref PubMed Scopus (468) Google Scholar) in which there are two substrate-binding sites where ubiquinol is oxidized (center P) and ubiquinone is re-reduced (center N). Crystal structures of the bc1 complexes obtained from various sources (2Xia D. Yu C.A. Kim H. Xian J.Z. Kachurin A.M. Zhang L. Yu L. Deisenhofer J. Science. 1997; 277: 60-66Crossref PubMed Scopus (867) Google Scholar, 3Zhang Z.L. Huang L.S. Shulmeister V.M. Chi Y.I. Kim K.K. Hung L.W. Crofts A.R. Berry E.A. Kim S.H. Nature. 1998; 392: 677-684Crossref PubMed Scopus (929) Google Scholar, 4Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure Fold Des. 2000; 8: 669-684Abstract Full Text Full Text PDF Scopus (508) Google Scholar) show a dimeric structure in which the two iron-sulfur protein subunits span both monomers in an intertwined arrangement. Crystal structures from the yeast bc1 complex show ubiquinone at center N of only one monomer and cytochrome c also in only one monomer, suggesting a functional asymmetry in the dimer (5Lange C. Hunte C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2800-2805Crossref PubMed Scopus (306) Google Scholar).Some center P inhibitors have been shown to completely block bc1 complex activity upon binding to only half of the dimeric complex (6Gutierrez-Cirlos E.B. Trumpower B.L. J. Biol. Chem. 2002; 277: 1195-1202Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), suggesting anti-cooperative interaction between the ubiquinol oxidation sites in the dimer. Antimycin, a center N inhibitor, binds to only one center N of the dimeric enzyme in a mutant where the iron-sulfur cluster cannot be inserted into the Rieske protein, suggesting conformational interaction between centers P and N of different monomers (7Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2002; 277: 50703-50709Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). In addition, it has long been observed that titration of the cytochrome c reductase activity of the enzyme with antimycin yields non-linear curves (8Berden J.A. Slater E.C. Biochim. Biophys. Acta. 1970; 216: 237-249Crossref PubMed Scopus (151) Google Scholar, 9Slater E.C. Biochim. Biophys. Acta. 1973; 301: 129-154Crossref PubMed Scopus (203) Google Scholar), which is unexpected for a tightly bound inhibitor. This anomalous behavior has been attributed to rapid mobility of the inhibitor between the two center N sites in the dimer (10Bechmann G. Weiss H. Rich P.R. Eur. J. Biochem. 1992; 208: 315-325Crossref PubMed Scopus (29) Google Scholar), although an alternate explanation could be equilibration of electrons between the cytochrome b subunits (6Gutierrez-Cirlos E.B. Trumpower B.L. J. Biol. Chem. 2002; 277: 1195-1202Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar).In this work, we have analyzed the pre-steady and steady state kinetics of the isolated yeast bc1 complex in the presence of antimycin under conditions where the simultaneous detection of both electrons coming from the first ubiquinol oxidation at center P is possible. The results show that the number of center N sites blocked in the dimer determines whether ubiquinol oxidation can occur in one or both of the monomers, and that electron communication exists between cytochrome b subunits of the two monomers.EXPERIMENTAL PROCEDURESMaterials—Yeast nitrogen base (YNB) 1The abbreviations used are: YNB, yeast nitrogen base; DBH2, decyl-ubiquinol. without amino acids and with ammonium sulfate was obtained from U.S. Biological. Amino acids were from Aldrich, Sigma, Fluka, and ICN. Dodecylmaltoside was obtained from Roche Applied Science. DEAE-Bio-Gel was obtained from Bio-Rad. Antimycin, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, horse heart cytochrome c, decyl-ubiquinone, sodium ascorbate, and dithionite were purchased from Sigma. Decyl-ubiquinol was prepared as described (11Trumpower B.L. Edwards C.A. J. Biol. Chem. 1979; 254: 8697-8706Abstract Full Text PDF PubMed Google Scholar). Antimycin and decyl-ubiquinol were quantified by UV-spectroscopy (6Gutierrez-Cirlos E.B. Trumpower B.L. J. Biol. Chem. 2002; 277: 1195-1202Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) using extinction coefficients of 4.8 mm-1 cm-1 at 320 nm (12Von Jagow G. Link T.A. Methods Enzymol. 1986; 126: 253-271Crossref PubMed Scopus (312) Google Scholar) and 4.14 mm-1 cm-1 at 289 nm (13Rich P.R. Biochim. Biophys. Acta. 1984; 768: 53-79Crossref PubMed Scopus (315) Google Scholar), respectively.Purification of Cytochrome bc1 Complexes—Wild-type bc1 complex was isolated from Red Star cake yeast as described previously (14Ljungdahl P.O. Pennoyer J.D. Robertson D.E. Trumpower B.L. Biochim. Biophys. Acta. 1987; 891: 227-241Crossref PubMed Scopus (125) Google Scholar, 15Snyder C.H. Trumpower B.L. Biochim. Biophys. Acta. 1998; 1365: 125-134Crossref PubMed Scopus (44) Google Scholar). The bc1 complex with a Y185F mutation in the iron-sulfur protein was isolated from the iron-sulfur protein deletion strain JPJ1 (16Beckmann J.D. Ljungdahl P.O. Trumpower B.L. J. Biol. Chem. 1989; 264: 3713-3722Abstract Full Text PDF PubMed Google Scholar) complemented with the single-copy plasmid pEDRIP1-Y185F encoding iron-sulfur protein with the Y185F mutation (17Denke E. Merbitz-Zahradnik T. Hatzfeld O.M. Snyder C.H. Link T.A. Trumpower B.L. J. Biol. Chem. 1998; 273: 9085-9093Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). This strain was grown on synthetic dropout medium containing 0.7% YNB, 0.14% amino acid mix lacking tryptophan, and 2% dextrose as previously described (18Merbitz-Zahradnik T. Zwicker K. Nett J.H. Link T.A. Trumpower B.L. Biochemistry. 2003; 42: 13637-13645Crossref PubMed Scopus (34) Google Scholar). The bc1 complex was purified by the same procedure as used with the wild-type enzyme (14Ljungdahl P.O. Pennoyer J.D. Robertson D.E. Trumpower B.L. Biochim. Biophys. Acta. 1987; 891: 227-241Crossref PubMed Scopus (125) Google Scholar, 15Snyder C.H. Trumpower B.L. Biochim. Biophys. Acta. 1998; 1365: 125-134Crossref PubMed Scopus (44) Google Scholar). Quantification of the bc1 complexes was performed as described previously (6Gutierrez-Cirlos E.B. Trumpower B.L. J. Biol. Chem. 2002; 277: 1195-1202Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), using extinction coefficients of 17.5 mm-1 cm-1 at 553–539 for cytochrome c (19Yu C.A. Yu L. King T.E. J. Biol. Chem. 1972; 247: 1012-1019Abstract Full Text PDF PubMed Google Scholar) and 25.6 mm-1 cm-1 at 563–578 for cytochrome b heme (8Berden J.A. Slater E.C. Biochim. Biophys. Acta. 1970; 216: 237-249Crossref PubMed Scopus (151) Google Scholar).Pre-steady State Reduction of bc1 Complexes—Pre-steady state reduction of the cytochrome bc1 complexes was followed at room temperature by stopped flow rapid scanning spectroscopy using the OLIS Rapid Scanning Monochromator as described before (15Snyder C.H. Trumpower B.L. Biochim. Biophys. Acta. 1998; 1365: 125-134Crossref PubMed Scopus (44) Google Scholar). When using the wild-type bc1 complex, reactions were started by rapid mixing of 3 μm enzyme in assay buffer containing 50 mm Tris-HCl, pH 8.8, 250 mm sucrose, 1 mm sodium azide, 0.2 mm EDTA, and 0.05% Tween 20 against an equal volume of the same buffer containing different concentrations of decyl-ubiquinol. For measurements using the bc1 complex with the Y185F mutation in the iron-sulfur protein, Tris was replaced by 50 mm potassium phosphate, pH 7.0, in the assay medium.For each experiment, 6–8 data sets were averaged, and the oxidized spectrum was subtracted. The time course of absorbance change at 539, 554, 563, and 578 nm was extracted using software from OLIS and exported to the Origin 5.0 program (OriginLab Corp.). By comparing the dithionite and ascorbate-reduced spectra of the yeast bc1 complex, it was determined that cytochrome b reduction results in a 30% increase in the absorbance change at 554–539 nm used for monitoring cytochrome c1 reduction, as shown in Fig. 1. A similar value was obtained by deconvoluting the dithionite-reduced spectra into two components by fitting to a double Lorentzian function (not shown). Considering that the final concentration of cytochrome c1 in the pre-steady state experiments was 1.5 μm and that the path length of the mixing chamber is 2 cm, complete reduction of cytochrome b would induce an extra 0.016 absorbance change at 554–539. Therefore, since the absorbance at 563 nm of fully reduced cytochrome b would be 0.154, a correction factor of (ΔA 563–578 nm/0.154) × 0.016 was subtracted from ΔA 554–539 nm at each time point to obtain the absorbance change attributable to cytochrome c1. The contribution of cytochrome c1 at the wavelengths used for cytochrome b calculation (563–579 nm) was considered to be negligible from a comparison of the dithionite-reduced and the dithionite minus ascorbate-reduced spectra (Fig. 1). The absorbance changes of cytochrome c1 and b were fitted to either a monophasic or a biphasic exponential function in the Origin program.Pre-steady State Reduction of Exogenous Cytochrome c—The same procedure was followed as described above for the isolated bc1 complex except that 18 μm horse heart cytochrome c and 1 mm KCN were added to the assay buffer containing the bc1 complex before rapid mixing. The time course of absorbance change at 550–539 nm was not corrected for cytochrome b reduction as judged by the negligible contribution of the b hemes at this wavelength pair (see Fig. 1). The absorbances for cytochrome c and b were fitted in the Origin program to a monophasic or biphasic exponential function. Cytochrome c reduction was quantified using an extinction coefficient of 21.5 mm-1 cm-1 at 550–539 nm (20Margoliash E. Walasek O.F. Methods Enzymol. 1967; 10: 339-348Crossref Scopus (229) Google Scholar).Ubiquinol-Cytochrome c Reductase Activity Assays—Ubiquinol-cytochrome c reductase activity was measured by stopped flow spectroscopy using the OLIS Rapid Scanning Monochromator as described elsewhere (6Gutierrez-Cirlos E.B. Trumpower B.L. J. Biol. Chem. 2002; 277: 1195-1202Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Antimycin was added incrementally to a 1 mm bc1 complex solution in assay buffer containing 50 mm phosphate, pH 7.0, 250 mm sucrose, 1 mm sodium azide, 0.2 mm EDTA, and 0.05% Tween 20 at 4°. After 4 min, the antimycin-bc1 complex mixture was diluted to a concentration of 100 nm of enzyme in assay buffer at room temperature with 1 mm potassium cyanide and 40 μm decyl-ubiquinol. After two more minutes, the reaction was started by mixing against an equal volume of 60 μm cytochrome c in assay buffer. The non-enzymatic rate of reduction of cytochrome c by decyl-ubiquinol was obtained by mixing equal volumes of 60 μm cytochrome c in assay buffer against 40 μm decyl-ubiquinol in assay buffer, and was found to be negligible. The reaction was followed for 2 s. For each inhibitor concentration, six data sets were obtained, and the time course of cytochrome c reduction was extracted using the OLIS software. The rate of cytochrome c reduction was calculated from the absorbance increase at 550–539 nm, using an extinction coefficient of 21.5 mm-1 cm-1 (20Margoliash E. Walasek O.F. Methods Enzymol. 1967; 10: 339-348Crossref Scopus (229) Google Scholar).Kinetic Modeling—For simulations of pre-steady state reduction of cytochrome c1, cytochrome b, and exogenous cytochrome c, the Dynafit program (Biokin Ltd.) was used (21Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1339) Google Scholar). Dynafit allows simulation and fitting of either the initial velocities or the time course of enzyme reactions to different molecular mechanisms represented symbolically by a set of chemical equations. Rate constants and concentrations of enzyme and ligands can be allowed to vary during iterative fitting in order to obtain adjusted values. The scripts used for simulations are provided as Supplemental Data.Forward and reverse rate constants were assigned to each equation representing a reversible partial reaction. The slowest rate constant was assigned to the ubiquinol oxidation reaction at center P, while all other constants were assumed to be several orders of magnitude faster (17Denke E. Merbitz-Zahradnik T. Hatzfeld O.M. Snyder C.H. Link T.A. Trumpower B.L. J. Biol. Chem. 1998; 273: 9085-9093Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). This rate constant was determined by iterative fitting allowing only this parameter along with the off rate constants for decyl-ubiquinol binding to vary. Although the precise values of the individual non-rate-limiting constants are irrelevant to the result of the simulation or fitting, the ratio of the reverse/forward rates was chosen to correspond to the equilibrium constants calculated to exist under the experimental conditions used.For experiments with wild-type bc1 complex at pH 8.8 in the presence of excess antimycin, the redox potential (Em) of decyl-ubiquinol was calculated to be -16 mV assuming a ΔEm of 59 mV/pH unit with respect to an Em7 of 90 mV. The iron-sulfur protein Em was calculated to be 200 mV using the known ΔEm of the Rieske cluster as a function of pH (22Link T.A. Hagen W.R. Pierik A.J. Assmann C. von Jagow G. Eur. J. Biochem. 1992; 208: 685-691Crossref PubMed Scopus (103) Google Scholar), taking as a reference an Em7 of 285 mV (23Tsai A.L. Palmer G. Biochim. Biophys. Acta. 1983; 722: 349-363Crossref PubMed Scopus (53) Google Scholar). Assuming a pH-independent Em of 65 mV for bH (23Tsai A.L. Palmer G. Biochim. Biophys. Acta. 1983; 722: 349-363Crossref PubMed Scopus (53) Google Scholar), an average Em value of 132.5 mV is obtained for the electron acceptors in a concerted ubiquinol oxidation reaction (the electron transfer between bL and bH was obviated to simplify the mechanism). With a ΔEm of 148.5 mV between decyl-ubiquinol and the redox acceptors, an equilibrium constant of 18 was calculated for the first turnover at center P according to the Nernst equation. Using a Em of -20 mV for heme bL (23Tsai A.L. Palmer G. Biochim. Biophys. Acta. 1983; 722: 349-363Crossref PubMed Scopus (53) Google Scholar), Keq for the second turnover at center P is 8 in the presence of antimycin and assuming no electron equilibration between monomers. For simulations with the bc1 complex with a Y185F mutation in the iron-sulfur protein, Em7 values of 215 mV for iron-sulfur protein (17Denke E. Merbitz-Zahradnik T. Hatzfeld O.M. Snyder C.H. Link T.A. Trumpower B.L. J. Biol. Chem. 1998; 273: 9085-9093Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) and of 90 mV for decyl-ubiquinol were used, yielding Keq values of 2.2 and 1 for the first and second turnovers at center P without electron crossover between cytochrome b subunits. For electron transfer between iron-sulfur protein and cytochrome c1 (Em = 270 mV, Ref. 23Tsai A.L. Palmer G. Biochim. Biophys. Acta. 1983; 722: 349-363Crossref PubMed Scopus (53) Google Scholar), a Keq of 4 was used. Electron transfer between cytochrome c1 and horse heart cytochrome c was assumed to occur with Keq = 1.For decyl-ubiquinol and decyl-ubiquinone binding, the on rate constants were fixed arbitrarily to the same value. Iterative fitting allowing the off rate constants to vary while all other parameters were fixed resulted in 2-fold higher values for decyl-ubiquinol than for decyl-ubiquinone. For simulations in the presence of exogenous cytochrome c, extra steps were included in the mechanism to account for a slow antimycin insensitive rate of cytochrome c reduction. The reactions included assume the formation of semiquinone upon reduction of iron-sulfur protein, dissociation of the semiquinone and dismutation to decyl-ubiquinone. The rate of dismutation was arbitrarily fixed to a very high rate, and the reverse reaction of semiquinone formation at center P was also set to yield a Keq of 0.001.The rate constant for semiquinone dissociation from center P was then determined by iterative fitting. The values obtained were ∼4 times lower than the off-rate constant for decyl-ubiquinone. However, the fitted dissociation rate constant for semiquinone became larger if Keq for semiquinone formation was set to an even lower value, or if the dismutation rate was increased. Therefore, the actual affinity for semiquinone at center P cannot be determined independently of the two other parameters that influence the stability of semiquinone. Nevertheless, the chosen parameters resulted in good fitting of the antimycin-insensitive rate of cytochrome c reduction. For cytochrome c binding, a Kd of 30 μm was chosen, and the individual on and off rate constants were arbitrarily set.Simulation of the relative concentrations of free enzyme and enzyme-inhibitor complexes as a function of antimycin/bc1 complex ratios was performed in Dynafit assuming no cooperativity in the binding of the inhibitor to the second monomer in the dimer. The Kd for antimycin was arbitrarily set to 0.2 nm, based on apparently stoichiometric binding of the inhibitor to the yeast bc1 complex in assays with the enzyme concentration at 2 nm (6Gutierrez-Cirlos E.B. Trumpower B.L. J. Biol. Chem. 2002; 277: 1195-1202Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The initial rates of cytochrome c reduction as a function of antimycin concentration were also fitted to different models using Dynafit scripts included as Supplemental Data.RESULTSPre-steady State Reduction of Cytochrome c1 by Decyl-ubiquinol in the Presence of Antimycin—At pH 7 the Em of the iron-sulfur protein in the bc1 complex from wild-type yeast is ∼15 mV higher than that of cytochrome c1. Consequently, oxidation of ubiquinol at center P does not result in more that 40% cytochrome c1 reduction during the first turnover of the enzyme, since the electron that is transferred from the quinol to the high potential acceptors, iron-sulfur protein, and cytochrome c1, equilibrates primarily to the former. However, at pH 8.8, the Em of the iron-sulfur protein is about 70 mV lower than that of cytochrome c1. Therefore, at pH 8.8 a first turnover of ubiquinol oxidation at center P should result in the reduction of ∼80% of the total content of cytochrome c1. However, in the presence of antimycin, as shown in Fig. 2, no more than 50% of the total cytochrome c1 underwent reduction even in the presence of 4 equivalents of decyl-ubiquinol. When one equivalent of substrate was available, only ∼40% of cytochrome c1 was reduced in a single phase, while kinetic simulations (dashed lines in Fig. 2) show that a 65% reduction level is expected. With 2 and 4 equivalents of decyl-ubiquinol, after the reduction of 40%, an additional 10% of c1 was reduced with a slower rate. In contrast, kinetic modeling showed that over 90% of c1 should be reduced, assuming that all of the bc1 complex present is active. Interestingly, the reduction levels of c1 could be accurately simulated assuming that only half of the bc1 complex is participating in decyl-ubiquinol oxidation (solid lines in Fig. 2).Fig. 2Pre-steady state reduction kinetics of cytochrome c1 by decyl-ubiquinol in cytochrome bc1 complex from wild-type yeast in the presence and absence of antimycin at pH 8.8. The bc1 complex was suspended at 1.5 μm and reduced with 0.75 μm (A), 1.5 μm (B), 3 μm (C), and 6 μm (D) decyl-ubiquinol. Antimycin (anti) was present at a concentration of 3 μm where indicated. Traces show cytochrome c1 absorbance at 554–539 nm after correction for cytochrome b spectral overlap. Rates and reduction extent (%) are indicated for each trace. Absorbance changes corresponding to 50 and 100% reduction and calculated for a path length of 2 cm are shown as horizontal bars. Simulated cytochrome c1 reduction kinetics assuming a concentration of active enzyme of 0.75 μm (solid curves) or 1.5 μm (dashed curves) are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the absence of antimycin, up to 80% reduction of c1 is observed with 2 equivalents of decyl-ubiquinol (Fig. 2C), indicating that the proportion of permanently inactive bc1 complex due to denaturation is not more than 20%. The fact that complete reduction of c1 was not observed at any decyl-ubiquinol concentration is probably due to decyl-ubiquinol binding to center N, thereby reducing heme bH or blocking its reoxidation. This was confirmed by monitoring the reduction level of cytochrome b in the absence of antimycin, which showed a complete inhibition of the reoxidation phase at concentrations above 2 equivalents of decyl-ubiquinol (data not shown).Even in the presence of a 10-fold molar excess of decyl-ubiquinol, only 55% of cytochrome c1 was reduced after several minutes in the presence of antimycin (Fig. 3B), in contrast with the more than 90% reduction level reached in the absence of the inhibitor (Fig. 3A). The kinetics of c1 reduction without antimycin showed that most of this cytochrome c1 was reduced in the second slow phase (data not shown), indicating that inhibition of cytochrome b reoxidation at center N by decyl-ubiquinol becomes considerable at this high substrate concentration.Fig. 3Spectra of cytochrome bc1 complex reduced by decyl-ubiquinol in the absence or presence of antimycin. Cytochrome bc1 complex from wild-type yeast was suspended at 1.5 μm in assay buffer at pH 8.8 and reduced with 15 μm decyl-ubiquinol in the stopped flow mixing chamber in the absence (A) or presence (B) of 3 μm antimycin, and spectra were recorded after 2 min. A spectrum of the same amount of oxidized bc1 complex mixed with buffer lacking decyl-ubiquinol was subtracted. The absorbance in the reduced-oxidized spectra (solid lines) was normalized to zero at the reference wavelengths of 539 nm and 579 nm before deconvolution to obtain cytochrome c1 (dashed curve) and cytochrome b (dotted curve) spectral contributions using a Lorentzian function with two components.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Similar results were obtained using the bc1 complex with a Y185F mutation in the iron-sulfur protein (Fig. 4). The iron-sulfur protein Em in this bc1 complex has a value of 215 mV at pH 7 (17Denke E. Merbitz-Zahradnik T. Hatzfeld O.M. Snyder C.H. Link T.A. Trumpower B.L. J. Biol. Chem. 1998; 273: 9085-9093Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), almost the same as that of the iron-sulfur protein in the wild-type enzyme at pH 8.8. Consequently, 80% of cytochrome c1 is expected to be reduced, assuming every center P is able to turn over once. This should occur with 2 or more equivalents of decyl-ubiquinol, according to kinetic modeling (Fig. 4, dashed lines). However, not even a 4-fold excess of substrate resulted in the reduction of more than ∼40% of the c1, in agreement with simulations that assume that only half of the bc1 complex is active in the presence of antimycin (solid lines). As in the case of the bc1 complex from wild-type yeast, over 80% of the enzyme with the Y185F mutation in the iron-sulfur protein was active in the absence of inhibitor, according to the level of c1 reduction with 1 and 2 equivalents of decyl-ubiquinol (Fig. 4, B and C). Since at pH 7 the Em of decyl-ubiquinol is over 100 mV higher than at pH 8.8, reduction of heme bH by the quinol through center N was decreased (data not shown), resulting in a greater amount of c1 reduction in the bc1 complex with the Y185F iron-sulfur protein at pH 7.0 at low substrate concentrations as compared with wild-type enzyme at pH 8.8. In addition, only monophasic reduction kinetics were observed in the presence of antimycin, indicating that only one turnover per active center P occurred with up to 4 substrate equivalents.Fig. 4Pre-steady state kinetics of cytochrome c1 reduction by decyl-ubiquinol in the presence and absence of antimycin at pH 7 in a bc1 complex with a Y185F iron-sulfur protein mutation. Cytochrome bc1 complex with a Y185F mutation in the iron-sulfur protein was suspended at 1.5 μm and reduced with 0.75 μm (A), 1.5 μm (B), 3 μm (C), and 6 μm (D) decyl-ubiquinol in the absence or presence of 3 μm antimycin (anti). The traces show cytochrome c1 absorbance at 554–539 nm after correction for cytochrome b spectral overlap. Rates and reduction extent (%) are indicated for each trace. Absorbance changes corresponding to 50 and 100% reduction and calculated for a path length of 2 cm are shown as horizontal bars. Simulated cytochrome c1 reduction kinetics assuming a concentration of active enzyme of 0.75 μm (solid curves) or 1.5 μm (dashed curves) are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Pre-steady State Reduction of Cytochrome b by Decyl-ubiquinol in the Presence of Antimycin—Given that for every electron transferred from ubiquinol to iron-sulfur protein and cytochrome c1 one electron is observable in cytochrome b, the extent of reduction of this cytochrome in the presence of antimycin can also indicate the relative proportion of active enzyme. Furthermore, the extinction coefficients of each of the two b hemes are different; bH reduction contributes 70% of the total absorbance change when cytochrome b is completely reduced, and bL the remaining 30% (24Rich P.R. Jeal A.E. Madgwick S.A. Moody A.J. Biochim. Biophys. Acta. 1990; 1018: 29-40Crossref PubMed Scopus (87) Google Scholar). The equilibrium constant of electron sharing between the two b hemes is largely displaced toward bH due to a ΔEm >90 mV between the two redox centers (23Tsai A.L. Palmer G. Biochim. Biophys. Acta. 1983; 722: 349-363Crossref PubMed Scopus (53) Google Scholar). This implies that when cytochrome b reoxidation through center N is prevented, the first ubiquinol oxidation in a monomer would reduce heme bH almost exclusively, and a second turnover would reduce bL. This makes it possible to distinguish spectroscopically between one monomer oxidizing two ubiquinol molecules at center P (resulting in 50% of the absorbance change that would result from total cytochrome b reduction) and two monomers turning over once (resulting in 70% of the maximum possible absorbance change) by using differe