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Regulatory Interactions between Ubiquinol Oxidation and Ubiquinone Reduction Sites in the Dimeric Cytochrome bc1 Complex

2006; Elsevier BV; Volume: 281; Issue: 41 Linguagem: Inglês

10.1074/jbc.m604694200

1083-351X

Raúl Covián, Bernard L. Trumpower,

Metal-Catalyzed Oxygenation Mechanisms

We have obtained evidence for conformational communication between ubiquinol oxidation (center P) and ubiquinone reduction (center N) sites of the yeast bc1 complex dimer by analyzing antimycin binding and heme bH reduction at center N in the presence of different center P inhibitors. When stigmatellin was occupying center P, concentration-dependent binding of antimycin occurred only to half of the center N sites. The remaining half of the bc1 complex bound antimycin with a slower rate that was independent of inhibitor concentration, indicating that a slow conformational change needed to occur before half of the enzyme could bind antimycin. In contrast, under conditions where the Rieske protein was not fixed proximal to heme bL at center P, all center N sites bound antimycin with fast and concentration-dependent kinetics. Additionally, the extent of fast cytochrome b reduction by menaquinol through center N in the presence of stigmatellin was approximately half of that observed when myxothiazol was bound at center P. The reduction kinetics of the bH heme by decylubiquinol in the presence of stigmatellin or myxothiazol were also consistent with a model in which fixation of the Rieske protein close to heme bL in both monomers allows rapid binding of ligands only to one center N. Decylubiquinol at high concentrations was able to abolish the biphasic binding of antimycin in the presence of stigmatellin but did not slow down antimycin binding rates. These results are discussed in terms of half-of-the-sites activity of the dimeric bc1 complex. We have obtained evidence for conformational communication between ubiquinol oxidation (center P) and ubiquinone reduction (center N) sites of the yeast bc1 complex dimer by analyzing antimycin binding and heme bH reduction at center N in the presence of different center P inhibitors. When stigmatellin was occupying center P, concentration-dependent binding of antimycin occurred only to half of the center N sites. The remaining half of the bc1 complex bound antimycin with a slower rate that was independent of inhibitor concentration, indicating that a slow conformational change needed to occur before half of the enzyme could bind antimycin. In contrast, under conditions where the Rieske protein was not fixed proximal to heme bL at center P, all center N sites bound antimycin with fast and concentration-dependent kinetics. Additionally, the extent of fast cytochrome b reduction by menaquinol through center N in the presence of stigmatellin was approximately half of that observed when myxothiazol was bound at center P. The reduction kinetics of the bH heme by decylubiquinol in the presence of stigmatellin or myxothiazol were also consistent with a model in which fixation of the Rieske protein close to heme bL in both monomers allows rapid binding of ligands only to one center N. Decylubiquinol at high concentrations was able to abolish the biphasic binding of antimycin in the presence of stigmatellin but did not slow down antimycin binding rates. These results are discussed in terms of half-of-the-sites activity of the dimeric bc1 complex. The cytochrome bc1 complex is a dimer of 9-11 subunits in mitochondria (1Xia 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 (868) Google Scholar, 2Zhang 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 (930) Google Scholar, 3Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure Fold Des. 2000; 8: 669-684Abstract Full Text Full Text PDF Scopus (509) Google Scholar) and 3-4 in bacteria (4Berry E.A. Huang L.S. Saechao L.K. Pon N.G. Valkova-Valchanova M. Daldal F. Photosynth. Res. 2004; 81: 251-275Crossref PubMed Scopus (175) Google Scholar) that catalyzes electron transfer from quinol (QH2) 2The abbreviations used are: QH2, quinol; MQ, menaquinone; MQH2, menaquinol (2,3-dimethyl-1,4-naphthoquinol); Q, quinone; SQ, semiquinone; DBH2, decylubiquinol (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol). 2The abbreviations used are: QH2, quinol; MQ, menaquinone; MQH2, menaquinol (2,3-dimethyl-1,4-naphthoquinol); Q, quinone; SQ, semiquinone; DBH2, decylubiquinol (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol). to cytochrome c coupled to proton movement across the membrane as described by the proton motive Q cycle model (5Mitchell P. J. Theor. Biol. 1976; 62: 327-367Crossref PubMed Scopus (922) Google Scholar, 6Trumpower B.L. Gennis R.B. Annu. Rev. Biochem. 1994; 63: 675-716Crossref PubMed Scopus (468) Google Scholar). The Rieske iron-sulfur protein of each monomer transports one electron from the QH2 oxidation site (center P) in cytochrome b to cytochrome c1. This electron shuttling by the Rieske protein requires a considerable movement of its extrinsic domain (7Darrouzet E. Valkova-Valchanova M. Moser C.C. Dutton P.L. Daldal F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4567-4572Crossref PubMed Scopus (137) Google Scholar, 8Nett J.H. Hunte C. Trumpower B.L. Eur. J. Biochem. 2000; 267: 5777-5782Crossref PubMed Scopus (65) Google Scholar, 9Xiao K. Yu L. Yu C.A. J. Biol. Chem. 2000; 275: 38597-38604Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The other electron from QH2 oxidation is transferred to the center N site in cytochrome b through the bL and bH hemes, where it reduces Q to form a stable, tightly bound semiquinone (SQ) (10Ohnishi T. Trumpower B.L. J. Biol. Chem. 1980; 255: 3278-3284Abstract Full Text PDF PubMed Google Scholar).When center N is blocked by inhibitors such as antimycin, center P starts to use oxygen as an electron acceptor when cytochrome b becomes reduced after a few turnovers, resulting in the formation of reactive oxygen species (11Kramer D.M. Roberts A.G. Muller F. Cape J. Bowman M.K. Methods Enzymol. 2004; 382: 21-45Crossref PubMed Scopus (45) Google Scholar). A similar situation could potentially arise when the Q pool is highly reduced, which would slow down the capacity of center N to reoxidize the b hemes by binding and reducing Q. We have recently provided evidence for mechanisms that maximize the availability of electron acceptors in cytochrome b in order to prevent reduction of oxygen at center P. We have found that only one center P in the dimer is active when both center N sites are occupied by antimycin (12Covian R. Gutierrez-Cirlos E.B. Trumpower B.L. J. Biol. Chem. 2004; 279: 15040-15049Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and that electrons coming from that QH2 oxidation site can equilibrate to any of the two bH hemes in the dimer by means of rapid bL-bL electron transfer (13Covian R. Trumpower B.L. J. Biol. Chem. 2005; 280: 22732-22740Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar).According to these previous findings, there would be four b hemes available to accept electrons from the one active center P site in the dimer. Under uninhibited conditions, electron equilibration between the cytochrome b subunits would favor the formation of SQ with oxidized bH heme at each center N, adding two more electron acceptors per active center P. Our kinetic analysis also suggested that both center P sites can be active simultaneously when only one center N is inhibited by antimycin (12Covian R. Gutierrez-Cirlos E.B. Trumpower B.L. J. Biol. Chem. 2004; 279: 15040-15049Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), hinting at possible conformational communication between center P and center N in the dimer. Antimycin is known to increase the susceptibility of the extrinsic domain of the Rieske protein to proteolysis (14Valkova-Valchanova M. Darrouzet E. Moomaw C.R. Slaughter C.A. Daldal F. Biochemistry. 2000; 39: 15484-15492Crossref PubMed Scopus (39) Google Scholar), suggesting that occupancy at center N affects its mobility. It has also been found recently that inhibitor binding, as well as mutations at center N, modify the interaction of the Rieske extrinsic domain with center P occupants (15Cooley J.W. Ohnishi T. Daldal F. Biochemistry. 2005; 44: 10520-10532Crossref PubMed Scopus (54) Google Scholar). However, the role of center P-center N conformational communication in regulating the half-of-the-sites-reactivity of the dimeric bc1 complex has not been explored.In the present work, we have demonstrated that occupancy of both center P sites by inhibitors that have opposite effects on the mobility of the extrinsic domain of the Rieske protein results in different kinetics of antimycin binding and QH2 oxidation at center N. Our results support a model in which interaction of both Rieske proteins with center P ligands largely inactivates one center N in the dimer, implying conformational communication not only between center P and center N but also between center N sites. We discuss these findings as part of a mechanism that allows only one center P in the dimer to be active at a time.EXPERIMENTAL PROCEDURESMaterials—Dodecylmaltoside was obtained from Roche Applied Science. DEAE-Bio-Gel was obtained from Bio-Rad Laboratories. Stigmatellin was from Fluka. Antimycin, myxothiazol, diisopropyl fluorophosphate, horse heart cytochrome c, decylubiquinone, sodium ascorbate, sodium dithionite, and sodium borohydride were purchased from Sigma. Menaquinone (MQ) was synthesized in the laboratory. DBH2 and MQH2 were prepared as described before (16Trumpower B.L. Edwards C.A. J. Biol. Chem. 1979; 254: 8697-8706Abstract Full Text PDF PubMed Google Scholar, 17Snyder C.H. Trumpower B.L. Biochim. Biophys. Acta. 1998; 1365: 125-134Crossref PubMed Scopus (44) Google Scholar). Antimycin, myxothiazol, stigmatellin, and DBH2 were quantified by UV spectroscopy (18Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2002; 277: 1195-1202Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) using reported extinction coefficients (19Von Jagow G. Link T.A. Methods Enzymol. 1986; 126: 253-271Crossref PubMed Scopus (312) Google Scholar, 20Rich P.R. Biochim. Biophys. Acta. 1984; 768: 53-79Crossref PubMed Scopus (315) Google Scholar). MQH2 was quantified by determining the amount of cytochrome c reduced by 50 nm isolated bc1 complex, assuming a ratio of 2 cytochromes c reduced/MQH2 oxidized.Purification of Cytochrome bc1 Complex—Wild-type bc1 complex was isolated from Red Star cake yeast as described previously (16Trumpower B.L. Edwards C.A. J. Biol. Chem. 1979; 254: 8697-8706Abstract Full Text PDF PubMed Google Scholar, 21Ljungdahl P.O. Pennoyer J.D. Robertson D.E. Trumpower B.L. Biochim. Biophys. Acta. 1987; 891: 227-241Crossref PubMed Scopus (125) Google Scholar). Quantification of the bc1 complex was performed as reported before (17Snyder C.H. Trumpower B.L. Biochim. Biophys. Acta. 1998; 1365: 125-134Crossref PubMed Scopus (44) Google Scholar), using extinction coefficients of 17.5 mm-1 cm-1 at 553-539 for cytochrome c1 (22Yu 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 562-579 for the average absorbance of the bH and bL hemes in cytochrome b (23Berden J.A. Slater E.C. Biochim. Biophys. Acta. 1970; 216: 237-249Crossref PubMed Scopus (151) Google Scholar). The amount of endogenous Q copurified with the bc1 complex was determined as described before (13Covian R. Trumpower B.L. J. Biol. Chem. 2005; 280: 22732-22740Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and determined to be 1.0-1.2 molecules/bc1 monomer.Kinetics of Antimycin Binding to the bc1 Complex—The appearance of the red shift of the reduced spectrum of the bH heme upon antimycin binding was followed at 20 °C by stopped flow rapid scanning spectroscopy using the OLIS Rapid Scanning Monochromator as previously reported (13Covian R. Trumpower B.L. J. Biol. Chem. 2005; 280: 22732-22740Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Purified yeast bc1 complex (5-6 μm) in assay buffer containing 50 mm phosphate, pH 7.0, 1 mm sodium azide, 0.2 mm EDTA, 0.05% Tween 20, and, where indicated, 1.2 equivalents/bc1 complex monomer of stigmatellin or myxothiazol and varying concentrations of DBH2 were reduced with a few grains of solid dithionite and mixed rapidly against an equal volume of the same buffer (without enzyme or center P inhibitors) containing different concentrations of antimycin. For each experiment, eight to ten data sets were averaged and the reduced spectrum was subtracted. The wavelength maximum and minimum for the antimycin-induced red shift (565 and 559 nm, respectively) were extracted using software from OLIS. The difference between the two wavelengths was plotted and fitted to a first or second order exponential equation using the Origin 5.0 (OriginLab Corp.) program.Pre-steady State Reduction of bc1 Complex—Pre-steady state reduction of cytochrome b was followed at 20 °C by stopped flow rapid scanning spectroscopy using the OLIS Rapid Scanning Monochromator as described before (17Snyder C.H. Trumpower B.L. Biochim. Biophys. Acta. 1998; 1365: 125-134Crossref PubMed Scopus (44) Google Scholar). Reactions were started by rapid mixing of 3 μm enzyme (expressed as monomers of bc1 complex) in the same assay buffer used for the antimycin binding experiments containing 3.6 μm stigmatellin or myxothiazol against an equal volume of the same buffer (without enzyme and inhibitors) containing different concentrations of MQH2 or DBH2. For each experiment, eight to ten data sets were averaged and the oxidized spectrum was subtracted. The time course of absorbance change at 562 and 578 nm was extracted using software from OLIS. The difference between the two wavelengths was plotted and fitted to a second or third order exponential equation using the Origin program.Kinetic Modeling—The Dynafit program (Biokin, Ltd.) calculates and solves a system of differential equations that correspond to the time-dependent change in concentration for each species involved in a given reaction mechanism, including substrates and products as well as any other ligands (24Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1339) Google Scholar). The mechanism is described as a series of individual reaction steps. A family of kinetic traces where one or more ligand concentrations are changed can be fitted globally to one or more kinetic models. An extinction coefficient can be assigned to some of those species that contribute to observed absorbance changes.Two models were used to fit the cytochrome b reduction kinetics by DBH2. For simplicity, only bH was assumed to undergo reduction, and an extinction coefficient of 36 mm-1 cm-1 was used for this heme group (12Covian R. Gutierrez-Cirlos E.B. Trumpower B.L. J. Biol. Chem. 2004; 279: 15040-15049Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 13Covian R. Trumpower B.L. J. Biol. Chem. 2005; 280: 22732-22740Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In addition, association and dissociation of DBH2, decylubiquinone, QH2, and Q were included together with the corresponding electron transfer reactions into single steps, thereby reducing the number of intermediate species. SQ species formed from partial reduction or oxidation of DBH2 or endogenous Q were assumed not to dissociate from the enzyme. Electron equilibration between the two bH hemes in the dimer through the bL hemes (13Covian R. Trumpower B.L. J. Biol. Chem. 2005; 280: 22732-22740Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) was described as a single step. One model assumed that all ligands were able to bind and react with both bH hemes in the dimer with identical rate constants, whereas the second model considered only one center N to be accessible initially, with the second center N being slowly and irreversibly activated by reaction at the first center N. The complete Dynafit script files are available as supplemental data.RESULTSBinding of Antimycin to Center N in the Presence or Absence of Center P Inhibitors—The dithionite-reduced bc1 complex bound antimycin at the vicinity of the bH heme with the kinetics shown in Fig. 1. The absorbance red shift induced by antimycin binding when center P was unoccupied or when myxothiazol was occupying center P occurred with monophasic kinetics (Fig. 1A). In contrast, when stigmatellin was present at center N, only half the center N sites were occupied with a rate similar to that observed in the vacant or myxothiazol-inhibited center P (Fig. 1B). The remaining half of center N sites bound antimycin with markedly slower kinetics. The contribution of the fast and slow phase to the total absorbance red shift between different antimycin concentrations and enzyme preparations varied between 45 and 55%, averaging 50% overall. The calculated second order rate constants for the binding of antimycin with or without myxothiazol were practically identical to each other and to the fast binding of antimycin to the stigmatellin-bound enzyme (Fig. 2).FIGURE 2Concentration dependence of antimycin binding rates. The rates of appearance of the antimycin-induced red shift with no center P inhibitors (A, open circles), in the presence of myxothiazol (A, solid circles) or stigmatellin (B, solid circles, fast phase; open circles, slow phase) were plotted as a function of antimycin concentration. Each family of points was fitted to a straight line with the value of the slope corresponding to the second order rate constant of antimycin binding to center N. Center P inhibitors and bc1 complex concentrations were as in Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Interestingly, the slow binding phase appeared to be largely independent of the concentration of antimycin (Fig. 2B), suggesting that a process slower than the diffusion of antimycin to center N was delaying binding of this inhibitor to half of the bc1 complex monomers. Because only one equivalent of antimycin/bc1 complex monomer was still sufficient to achieve maximal red shift even in the presence of stigmatellin (data not shown), the slow antimycin binding to half of the center N sites was not accompanied by a significant loss in overall affinity toward the inhibitor. In these experiments, center P inhibitors were added at an almost stoichiometric ratio (1.2 equivalents/bc1 complex monomer), and given that the same red shift kinetics were observed with higher (2 or 3 equivalents) concentrations of stigmatellin or myxothiazol (data not shown), the observed differences in antimycin binding cannot be attributed to unspecific binding of the center P inhibitors at center N.Reduction of bH Heme by MQH2 in the Presence of Myxothiazol or Stigmatellin—To determine whether the center P inhibitor-dependent binding pattern of antimycin was relevant to the interaction of substrate at center N, the low redox potential substrate MQH2 (Em7 =-70 mV) was used to reduce cytochrome b through center N (Fig. 3). In the presence of myxothiazol, MQH2 reduced ∼90% of the bH hemes in a fast single phase, whereas stigmatellin bound at center P only allowed slightly over half of the bH hemes to undergo rapid reduction. In both cases a slower subsequent reduction was observed that resulted in some bL heme reduction when myxothiazol was present. In the stigmatellin-inhibited bc1 complex, less than half of the remaining oxidized bH heme was reduced during this slower phase. These results are consistent with the observed antimycin binding kinetics (Fig. 1) and indicate that half of the monomers are not able to bind MQH2 rapidly when stigmatellin is occupying center P. The fast reduction through center N had the same rate regardless of the center P inhibitor added, suggesting that the affinity for MQH2 binding and the stability of menasemiquinone (formed after the one electron oxidation of MQH2) are the same in all those center N sites that are able to react with the substrate (half of the total in the presence of stigmatellin or all of them with myxothiazol).FIGURE 3Cytochrome b reduction by MQH2 in the presence of center P inhibitors. Cytochrome bc1 complex (1.5 μm final) preincubated with 1.2 equivalents of myxothiazol (upper trace) or stigmatellin (lower trace) was reduced with 16 equivalents of MQH (24 μm final). Rate constants were calculated from fitting to a two-exponential function. The relative amount of bH reduced is indicated in parentheses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Reduction of bH Heme by DBH2 in the Presence of Myxothiazol or Stigmatellin—Because decylubiquinone has the same benzoquinone ring structure and redox potential (Em7 = 90 mV) as the natural Q substrate of the bc1 complex, DBH2 was used to compare the effect of myxothiazol and stigmatellin on center N reduction kinetics. As shown in Fig. 4A, ∼50% of the bH hemes were reduced by DBH2 in the presence of myxothiazol, mostly in a single fast phase.FIGURE 4Cytochrome b reduction by DBH2 in the presence of center P inhibitors. Cytochrome bc1 complex (1.5 μm) preincubated with 1.2 equivalents of myxothiazol (A) or stigmatellin/bc1 monomer (B) was reduced with 16 equivalents of MQH2 (24 μm). Rate constants were calculated from fitting to a two-exponential (A) or three-exponential (B) function. The relative amount of bH reduced is indicated in parentheses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In contrast, a more complicated kinetic pattern was evident when stigmatellin was bound at center P (Fig. 4B). In this case, ∼26% of the bH hemes were reduced with a rate similar to that of the fast phase of reduction observed with myxothiazol. An additional 24% was reduced with slower kinetics, followed by an even lower reoxidation rate that involved ∼6% of the total bH hemes. Once again, these results suggest that only half of the center N sites that react rapidly in the presence of myxothiazol are able to undergo rapid reduction when stigmatellin is bound at center P. A subsequent slow equilibration of electrons occurs once slower conformational changes allow the other half of the center N sites to bind the ∼1 equivalent of endogenous Q/monomer that is present in our purified bc1 complex preparations.To further test this possibility, we fitted the center N reduction kinetics at different DBH2 concentrations in the presence of stigmatellin to two different models of electron equilibration within the cytochrome b dimer (Fig. 5; see "Experimental Procedures" and supplemental data for detailed descriptions). When both center N sites were assumed to bind and react simultaneously with DBH2 and Q to form the corresponding SQ, a poor fitting to the experimentally measured rates was obtained (Fig. 5A). This model could not accurately account for the different extent of bH reduction at the various DBH2 concentrations and predicted a very weak reoxidation only at the highest substrate concentration. In contrast, good fitting was obtained using a model that assumed that center N in one monomer could not bind and react with any ligands until a slow irreversible conformational change occurred upon SQ formation in the active monomer (Fig. 5B).FIGURE 5Kinetic modeling of cytochrome b reduction by DBH2 in the presence of center P inhibitors. Cytochrome bc1 complex (1.5 μm) preincubated with 1.2 equivalents of stigmatellin (B) was reduced with 1, 2, 4, 8, 12, and 16 equivalents of DBH2/bc1 monomer. Traces were fitted to a model that assumed that both center N sites were equally active (A) or that one center N was inactive until formation of SQ at the active site induced a slow conformational change to activate the second center N site (B). Solid lines correspond to the fitted curves. See supplemental data for details on the kinetic models and fitted values for rate constants.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The fitted value for the rate of the intermonomeric conformational change (0.15 s-1) was one order of magnitude lower than the slow antimycin binding observed in the presence of stigmatellin (Figs. 1B and 2B), suggesting that the SQ formed from DBH2 oxidation is less efficient than antimycin in eliciting the activation of center N in the second monomer. Interestingly, cytochrome b reduction kinetics in the presence of myxothiazol could be fitted equally well to both models (see supplemental data). This occurred because the value for the rate of intermonomeric conformational change in the second model increased to very high, non-rate-limiting values, rendering this mechanism essentially equivalent to the first model in which both center N sites in the dimer are assumed to react simultaneously.Further proof that the active center N in the stigmatellin-inhibited bc1 dimer has the same kinetic properties as the center N sites in the myxothiazol-bound complex was obtained by calculating the affinities for DBH2 from the fast cytochrome b reduction kinetics in the presence of both inhibitors. As shown in Fig. 6, the Km values for DBH2 are similar irrespective of the center P inhibitor and the small difference can be attributed to the increased uncertainty in the rate of the fast bH reduction with stigmatellin due to the appearance of the reoxidation phase at the highest DBH2 concentrations.FIGURE 6Kinetic parameters of cytochrome b reduction by DBH2 in the presence of center P inhibitors. The rates of the first phase of bH reduction in the presence of 1.2 equivalents/bc1 monomer of myxothiazol (A) or stigmatellin (B) in an experiment similar to that shown in Fig. 5 are plotted as a function of DBH2 concentration. Data points were fitted to a hyperbolic function to obtain the indicated Km values for reduction by DBH2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of DBH2 on Antimycin Binding to the Center P Inhibited bc1 Complex—Antimycin binding rates were also determined after adding DBH2 to the dithionite-reduced bc1 complex bound with myxothiazol or stigmatellin (Fig. 6). Surprisingly, DBH2 did not decrease the single rate of antimycin binding in the presence of myxothiazol even at concentrations close to its solubility limit and one order of magnitude higher than its Km value in the oxidized bc1 complex (0.15 mm), even though antimycin was used at a low concentration of 2 equivalents/bc1 monomer (Fig. 7A). Likewise, in the presence of stigmatellin, the rate of the fast binding of antimycin to half of the center N sites was insensitive to DBH2 (Fig. 7B). However, DBH2 at the concentration shown did eliminate the second slow phase of antimycin binding, resulting in a single fast phase (Fig. 7B) similar to the kinetic pattern observed in the presence of myxothiazol. The disappearance of the slow phase was dependent on DBH2 concentration, with a Km similar to that obtained in the oxidized bc1 complex (data not shown).FIGURE 7Effect of DBH2 on the kinetics of antimycin binding to center N. The time course of the ferro-bH spectral red shift induced by 6 μm antimycin binding to 3 μm dithionite-reduced bc1 complex preincubated with (dotted traces) or without (solid traces) 150 μm DBH2 was determined in the presence of 3.6 μm myxothiazol (A) or stigmatellin (B). The indicated binding rates were obtained by fitting the kinetic traces to a first order function (after correcting for the change in free inhibitor concentration), with an additional phase in the case of the enzyme incubated with stigmatellin but without DBH2 (solid trace in panel B) accounting for ∼50% of the total red shift. Solid lines correspond to the fitted curves.View Large Image Figure ViewerDownload Hi-res image Download (PPT)This result could suggest that DBH2 does bind to the active center N in the reduced stigmatellin-bound enzyme, where it is able to trigger the slow conformational change that allows fast binding of antimycin to the second monomer. In this scenario, the lack of decrease in the observed fast binding rate of antimycin to the active center N sites would be due to binding of antimycin to a different form of the enzyme than that to which DBH2 binds. In this case, however, saturation with DBH2 would be expected to maintain the enzyme in a conformation different from that to which antimycin binds, resulting in a decreased apparent binding rate for the inhibitor. Therefore, it seems more likely that the reduced bc1 complex has a very low affinity toward DBH2 and that during the 1-2 min between the addition of DBH2 to the reduced enzyme and the moment of rapid mixing with antimycin the substrate was able to transiently bind to the active center N site in the dimer to promote the intermonomeric conformational change that activates the other center N. The high dissociation rate for the bc1-DBH2 complex, however, would prevent significant competition against antimycin, as evidenced by the lack of effect on the inhibitor binding rate to the