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• W2119055406 abstract "The cytochrome bc1 complex recycles one of the two electrons from quinol (QH2) oxidation at center P by reducing quinone (Q) at center N to semiquinone (SQ), which is bound tightly. We have analyzed the properties of SQ bound at center N of the yeast bc1 complex. The EPR-detectable signal, which reports SQ bound in the vicinity of reduced bH heme, was abolished by the center N inhibitors antimycin, funiculosin, and ilicicolin H, but was unchanged by the center P inhibitors myxothiazol and stigmatellin. After correcting for the EPR-silent SQ bound close to oxidized bH, we calculated a midpoint redox potential (Em) of ∼90 mV for all bound SQ. Considering the Em values for bH and free Q, this result indicates that center N preferentially stabilizes SQ·bH3+ complexes. This favors recycling of the electron coming from center P and also implies a >2.5-fold higher affinity for QH2 than for Q at center N, which would potentially inhibit bH oxidation by Q. Using pre-steady-state kinetics, we show that Q does not inhibit the initial rate of bH reduction by QH2 through center N, but does decrease the extent of reduction, indicating that Q binds only when bH is reduced, whereas QH2 binds when bH is oxidized. Kinetic modeling of these results suggests that formation of SQ at one center N in the dimer allows stabilization of SQ in the other monomer by Q reduction after intradimer electron transfer. This model allows maximum SQ·bH3+ formation without inhibition of Q binding by QH2. The cytochrome bc1 complex recycles one of the two electrons from quinol (QH2) oxidation at center P by reducing quinone (Q) at center N to semiquinone (SQ), which is bound tightly. We have analyzed the properties of SQ bound at center N of the yeast bc1 complex. The EPR-detectable signal, which reports SQ bound in the vicinity of reduced bH heme, was abolished by the center N inhibitors antimycin, funiculosin, and ilicicolin H, but was unchanged by the center P inhibitors myxothiazol and stigmatellin. After correcting for the EPR-silent SQ bound close to oxidized bH, we calculated a midpoint redox potential (Em) of ∼90 mV for all bound SQ. Considering the Em values for bH and free Q, this result indicates that center N preferentially stabilizes SQ·bH3+ complexes. This favors recycling of the electron coming from center P and also implies a >2.5-fold higher affinity for QH2 than for Q at center N, which would potentially inhibit bH oxidation by Q. Using pre-steady-state kinetics, we show that Q does not inhibit the initial rate of bH reduction by QH2 through center N, but does decrease the extent of reduction, indicating that Q binds only when bH is reduced, whereas QH2 binds when bH is oxidized. Kinetic modeling of these results suggests that formation of SQ at one center N in the dimer allows stabilization of SQ in the other monomer by Q reduction after intradimer electron transfer. This model allows maximum SQ·bH3+ formation without inhibition of Q binding by QH2. The cytochrome bc1 complex couples electron transfer from QH2 2The abbreviations used are: QH2, quinol; Q, quinone; SQ, semiquinone; DBQ, decylubiquinone; DBH2, decylubiquinol (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol); mT, milliteslas. to cytochrome c to a net movement of protons across the membrane in which it is embedded. This is achieved by having two QH2/Q-binding sites (center P and center N) in cytochrome b close to opposite sides of the membrane, as is clearly seen in crystallographic structures (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 (873) 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 (938) Google Scholar, 3Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure Folding Des. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 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 (176) Google Scholar). The bifurcated mechanism of QH2 oxidation at center P in the protonmotive Q cycle results in one of the electrons from the substrate being transferred to the bL heme and then to the bH heme, which is in close proximity to the center N-binding pocket. This electron is used to reduce Q, producing an SQ intermediate, which is further reduced to QH2 after a second oxidation event at center P. For proton translocation to occur, these two sites must function in opposite directions, so protons from QH2 oxidation at center P are released to the positive side of the membrane, whereas Q reduction at center N results in proton uptake from the negative side. However, the bH heme group responsible for Q reduction at center N has a midpoint redox potential at pH 7 (Em7) of 50–100 mV (5Dutton P.L. Jackson J.B. Eur. J. Biochem. 1972; 30: 495-510Crossref PubMed Scopus (160) Google Scholar, 6T'sai A.L. Palmer G. Biochim. Biophys. Acta. 1983; 722: 349-363Crossref PubMed Scopus (54) Google Scholar, 7Rich P.R. Jeal A.E. Madgwick S.A. Moody A.J. Biochim. Biophys. Acta. 1990; 1018: 29-40Crossref PubMed Scopus (88) Google Scholar), which is close to the value of 60–90 mV for the Q pool in the membrane (8Kroöger A. Klingenberg M. Biochim. Biophys. Acta. 1973; 34: 358-368Google Scholar, 9Takamiya K.I. Dutton P.L. Biochim. Biophys. Acta. 1979; 546: 1-16Crossref PubMed Scopus (99) Google Scholar). This implies that the bH heme should oxidize QH2 as easily as it can reduce Q. When electrons are prevented from flowing out of cytochrome b through center N, QH2 oxidation at center P is inhibited and results in detrimental side reactions, such as superoxide formation (10Kramer D.M. Roberts A.G. Muller F. Cape J. Bowman M.K. Methods Enzymol. 2004; 382: 21-45Crossref PubMed Scopus (45) Google Scholar, 11Muller F.L. Roberts A.G. Bowman M.K. Kramer D.M. Biochemistry. 2003; 42: 6493-6499Crossref PubMed Scopus (118) Google Scholar). We have recently provided evidence indicating fast electron equilibration between bH hemes in the bc1 complex dimer and suggested that this minimizes formation of inhibitory SQ·bH2+ complexes at center N (12Covian R. Trumpower B.L. J. Biol. Chem. 2005; 280: 22732-22740Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Furthermore, potentiometric studies of SQ bound at center N (13Ohnishi T. Trumpower B.L. J. Biol. Chem. 1980; 255: 3278-3284Abstract Full Text PDF PubMed Google Scholar, 14De Vries S. Berden J.A. Slater E.C. FEBS Lett. 1980; 122: 143-148Crossref PubMed Scopus (66) Google Scholar, 15Robertson D.E. Prince R.C. Bowyer J.R. Matsuura K. Dutton P.L. Ohnishi T. J. Biol. Chem. 1984; 259: 1758-1763Abstract Full Text PDF PubMed Google Scholar) have indicated that the Em of this intermediate is more positive than that of the Q pool by at least 20 mV, suggesting that formation of productive SQ·bH3+ complexes is favored by preferential binding of QH2 to center N. Nevertheless, this introduces another difficulty for optimal center N function because QH2 would prevent binding of Q to center N, especially at high QH2/Q ratios. We have analyzed the thermodynamic properties of bound SQ and the pre-steady-state kinetics of bH reduction through center N of the yeast bc1 complex. Our results point to a mechanism in which center N sites in the dimer selectively bind QH2 or Q depending on the redox state of the bH heme as well as on the occupancy of the other monomer. We discuss how this model maximizes SQ·bH3+ complex formation while preventing QH2 from interfering with Q binding. Materials—Dodecyl maltoside was obtained from Anatrace. Antimycin, myxothiazol, DBQ, and redox mediators were purchased from Sigma, except for menaquinone, which was synthesized in the laboratory. Funiculosin was a gift from Novartis (Basel, Switzerland), and ilicicolin H was from the Merck sample repository. DBH2 was prepared as described previously (16Trumpower B.L. Edwards C.A. J. Biol. Chem. 1979; 254: 8697-8706Abstract Full Text PDF PubMed Google Scholar). All inhibitors and DBH2 were quantified by UV spectroscopy (17Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2002; 277: 1195-1202Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) using previously reported extinction coefficients (18Von Jagow G. Link T.A. Method Enzymol. 1986; 126: 253-271Crossref PubMed Scopus (314) Google Scholar, 19Rich P.R. Biochim. Biophys. Acta. 1984; 768: 53-79Crossref PubMed Scopus (315) Google Scholar, 20Gutierrez-Cirlos E.B. Merbitz-Zahradnik T. Trumpower B.L. J. Biol. Chem. 2004; 279: 8708-8714Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Purification of Cytochrome bc1 Complex—Wild-type cytochrome bc1 complex was isolated from Red Star cake yeast as described previously (21Ljungdahl P.O. Pennoyer J.D. Robertson D.E. Trumpower B.L. Biochim. Biophys. Acta. 1987; 891: 227-241Crossref PubMed Scopus (126) Google Scholar). Quantification of the bc1 complex was performed as reported previously (22Snyder 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 nm for cytochrome c1 (23Yu 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 nm for the average absorbance of the bH and bL hemes in cytochrome b (24Berden 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 previously (12Covian R. Trumpower B.L. J. Biol. Chem. 2005; 280: 22732-22740Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) and varied between different preparations in the range of 0.8–1.2 molecules/bc1 monomer. Semiquinone Redox Titration—Purified bc1 complex was diluted with 100 mm Tris and 50 mm KCl (pH 7.4) to a final concentration of 24–30 μm (based on Rieske iron-sulfur cluster concentration as measured by EPR spectroscopy; see below). Upon addition of 33 μm 2,3,5,6-tetramethyl-p-phenylenediamine (Em7 =+270 mV) and 33 μm phenazine ethosulfate (Em7 = +55 mV) as redox mediators, the solution was transferred into an anaerobic vessel continuously flushed with argon. The appearance of additional artificial radical signals was avoided by using only two mediator dyes, one having its Em in the range of the expected value of the Rieske iron-sulfur cluster and the other in the range of the expected Em of SQ. The sample was stirred at a constant temperature of 298 K and poised at desired potential values by adding small aliquots of 50 mm sodium dithionite solution. The potential was monitored by a redox microelectrode (Mettler-Toledo GmbH, Gieβen, Germany), and at appropriate values, 100 μl of the solution were transferred into argon-flushed EPR tubes (4-mm diameter), frozen in a cold isopentane/methyl cyclohexane mixture (5:1, ∼120 K), and stored in liquid nitrogen until EPR measurements were taken. Usually, titrations started at potentials of +350 mV and were followed down to -120 mV. X-band EPR spectra were obtained with a Bruker ESP 300E spectrometer equipped with a Hewlett Packard HP 53159A frequency counter, a Bruker ER 035M NMR gaussmeter, and an Oxford Instruments liquid helium continuous flow cryostat. The degree of reduction of the Rieske iron-sulfur cluster was followed by recording EPR spectra at 20 K (microwave frequency, 9.47 GHz; microwave power, 2 milliwatts; modulation amplitude, 0.64 milliteslas (mT); and sweep width, 100 mT). The appearance of SQ signals was detected at 50 K (microwave frequency, 9.47 GHz; microwave power, 0.01 milliwatt; modulation amplitude, 0.2 mT; and sweep width, 5 mT). A Q-free bc1 preparation (25Snyder C.H. Trumpower B.L. J. Biol. Chem. 1999; 274: 31209-31216Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) was titrated in the same manner as the other samples to analyze the occurrence of any artificial radical signals or base-line drifts derived from mediator dyes or any other component in the solution. Although showing normal titration behavior of the Rieske iron-sulfur cluster, no EPR signal in the range of SQ radicals was detectable in this sample. For data analysis, all signal intensities of SQ radicals were normalized to the maximum intensity of the signal in the control titration, in the absence of any inhibitor. Quantitation of maximum SQ concentrations was achieved by accumulation of spectra at 50 K, subtraction of the spectrum of the Q-free sample, double integration, and comparison with spectra from galvinoxyl-free radical (Sigma) at known concentrations. For power saturation measurements, the modulation amplitude was decreased to 0.1 mT to avoid signal broadening, and the sweep time was doubled from 42 to 84 s. Saturation curves were fitted to a standard equation describing the behavior of one paramagnetic species (26Ohnishi T. Biochim. Biophys. Acta. 1998; 1364: 186-206Crossref PubMed Scopus (384) Google Scholar). Cytochrome b Redox Titration—Optical potentiometric titrations were performed at 24 °C in a 3.5-ml quartz cuvette as described previously (27Dutton P.L. Methods Enzymol. 1978; 54: 411-435Crossref PubMed Scopus (729) Google Scholar). The potential was measured with a platinum-Ag/AgCl (3M) microelectrode (MI-80414-6, Microelectrodes, Inc.). All values are expressed with respect to the normal hydrogen electrode. The electrode was calibrated against a pH 7 standard solution of quinhydrone (Em =+296 mV). The purified bc1 complex was diluted to 2 μm in 100 mm Tris (pH 7.4), 50 mm KCl, and 0.01% dodecyl maltoside. Redox equilibration between the protein and the electrode was achieved by a mixture of the following dyes (with their Em7 values): 70 μm 2,3,5,6-tetramethyl-p-phenylenediamine (+270 mV), 25 μm 1,2-naphthoquinone (+144 mV), 25 μm phenazine methosulfate (+80 mV), 25 μm phenazine ethosulfate (+55 mV), 50 μm duroquinone (+5 mV), 30 μm menaquinone (-76 mV), 50 μm 2-hydroxy-1,4-naphthoquinone (-145 mV), 30 μm anthraquinone 2,6-disulfonate (-184 mV), and 30 μm anthraquinone 2-sulfonate (-225 mV). A 10 or 100 mm solution of dithionite and ferricyanide was used for the reductive and oxidative titrations, respectively. The UV-visible spectra were recorded between 500 and 600 nm in an Aminco DW-2 dual-wavelength spectrophotometer in the split beam mode. The absorbance at 562 minus 578 nm was plotted against the potential (Eh) of the system. The reductive and oxidative titrations were averaged, and the resulting graph was fitted in the ORIGIN 5.0 program (OriginLab Corp.) to the following n = 1 Nernst equation (Equation 1) with two components to obtain the redox potential for the bH Em(bH) and bL (Em(bL) hemes as well as the relative contribution of the bH heme to the total absorbance (b), ΔA(562−578 nm)=Cɛp(bnFeRT(Em(bH)−Eh)1+nFeRT(Em(bH)−Eh)+(1−b)nFeRT(Em(bL)−Eh)1+nFeRT(Em(bL)−Eh))1 where C is the concentration of bc1 complex monomers (in this case, 2 μm), ɛ is the added extinction coefficient of both b hemes (51.2 mm-1 cm-1), and p is the light path length (in this case, 1 cm). The temperature of the assay was maintained at 24 °C. The Nernst plots for both oxidative and reductive titrations were essentially identical, indicating full reversibility in the titration and confirming that the system was in equilibrium. Pre-steady-state Reduction of Cytochrome b through Center N—Pre-steady-state reduction of cytochrome b was followed at 24 °C by stopped-flow rapid scanning spectroscopy using the OLIS rapid scanning monochromator as described previously (22Snyder 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 the bc1 complex) 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 eq of stigmatellin or myxothiazol/bc1 complex monomer and varying concentrations of DBQ against an equal volume of the same buffer (without enzyme and inhibitors) containing 48 μm DBH2. For each experiment, 12–16 data sets were averaged, and the oxidized spectrum was subtracted. The time course of the absorbance change at 562 and 578 nm was extracted using software from OLIS. Using the ORIGIN program, the difference between the two wavelengths was plotted and fitted to a second- or third-order exponential, and the fitted curve was then used as the basis for an iterative smoothing procedure to decrease the noise levels of the kinetic traces. In this procedure, the difference between each data point and the corresponding value of the fitted curve at the same time point was calculated and decreased by half. Kinetic Modeling—The DynaFit program (BioKin, Ltd.), which allows fitting to reaction mechanisms described as a series of individual reaction steps (28Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1357) Google Scholar), was used to fit the time-dependent bH reduction through center N. An extinction coefficient of 36 mm-1 cm-1 was used for this heme group (12Covian R. Trumpower B.L. J. Biol. Chem. 2005; 280: 22732-22740Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 29Covian R. Gutierrez-Cirlos E.B. Trumpower B.L. J. Biol. Chem. 2004; 279: 15040-15049Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Association and dissociation of DBH2, DBQ, 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 (12Covian R. Trumpower B.L. J. Biol. Chem. 2005; 280: 22732-22740Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) was described as a single step. For bH reduction in the myxothiazol-inhibited enzyme, the two models used assumed that all ligands were able to bind and react with both bH hemes in the dimer with identical rate constants. One of the models considered only one center N to be accessible initially, with the second center N being rapidly activated by SQ formation at the first site. When stigmatellin was present, the model used to fit the reduction kinetics assumed different kinetic parameters in one of the two center N sites in the dimer from the outset. The complete DynaFit script files are available as supplemental material. EPR Spectra of Semiquinone Bound at Center N—The purified yeast cytochrome bc1 complex has been reported to exhibit an EPR signal centered at g = 2 attributed to an SQ radical (6T'sai A.L. Palmer G. Biochim. Biophys. Acta. 1983; 722: 349-363Crossref PubMed Scopus (54) Google Scholar). However, that putative SQ signal was significantly different from SQ signals reported in other bc1 complexes (13Ohnishi T. Trumpower B.L. J. Biol. Chem. 1980; 255: 3278-3284Abstract Full Text PDF PubMed Google Scholar, 14De Vries S. Berden J.A. Slater E.C. FEBS Lett. 1980; 122: 143-148Crossref PubMed Scopus (66) Google Scholar, 15Robertson D.E. Prince R.C. Bowyer J.R. Matsuura K. Dutton P.L. Ohnishi T. J. Biol. Chem. 1984; 259: 1758-1763Abstract Full Text PDF PubMed Google Scholar) in that it had a much smaller intensity (only 5% of the Q content), yielded an Em for SQ that was ∼100 mV higher than in other organisms, and was pH-independent. No evidence was provided to demonstrate that such an EPR signal came from a center N radical. As shown in Fig. 1, we have now obtained an EPR signal (g = 2.004 and line width = 0.9 mT) that clearly corresponds to SQ bound at center N, as judged by its sensitivity to three different center N inhibitors (antimycin, funiculosin, and ilicicolin H) and by its absence in bc1 complex lacking Q. The maximum intensity of this SQ signal at a microwave power of 0.01 milliwatt varied between 0.06 and 0.27/bc1 complex monomer, depending on the Q content of the enzyme preparation, and occurred at an Em of 50–60 mV at pH 7.4, in agreement with what has been observed in other bc1 complexes (13Ohnishi T. Trumpower B.L. J. Biol. Chem. 1980; 255: 3278-3284Abstract Full Text PDF PubMed Google Scholar, 14De Vries S. Berden J.A. Slater E.C. FEBS Lett. 1980; 122: 143-148Crossref PubMed Scopus (66) Google Scholar, 15Robertson D.E. Prince R.C. Bowyer J.R. Matsuura K. Dutton P.L. Ohnishi T. J. Biol. Chem. 1984; 259: 1758-1763Abstract Full Text PDF PubMed Google Scholar). It was previously shown experimentally (30Siedow J.N. Power S. De la Rosa F.F. Palmer G. J. Biol. Chem. 1978; 253: 2392-2399Abstract Full Text PDF PubMed Google Scholar, 31De la Rosa F.F. Palmer G. FEBS Lett. 1982; 163: 140-143Crossref Scopus (33) Google Scholar) and confirmed theoretically (7Rich P.R. Jeal A.E. Madgwick S.A. Moody A.J. Biochim. Biophys. Acta. 1990; 1018: 29-40Crossref PubMed Scopus (88) Google Scholar) that the SQ bound at center N is anti-ferromagnetically coupled to the oxidized bH heme. Thus, to determine the true concentration and Em of SQ bound at center N, a correction needs to be made for the portion of the SQ that is EPR-silent due to this coupling. Because the reported Em of yeast cytochrome b using either circular dichroism or EPR spectroscopy was found to be dependent on the type of detergent present (6T'sai A.L. Palmer G. Biochim. Biophys. Acta. 1983; 722: 349-363Crossref PubMed Scopus (54) Google Scholar), we performed a spectrophotometric redox titration of the b hemes in the presence of dodecyl maltoside in the same buffer as that used for our EPR experiments. As shown in Fig. 2A, at pH 7.4, the bH heme has an Em of ∼60 mV. Using this value, we calculated the total percentage (relative to bc1 monomers) of SQ bound at all center N sites at different redox potential values SQtotEh from the g = 2.0 EPR signal, which reflects only SQ bound in the vicinity of bH2+ heme complexes SQbHEh2+, expressed as a percentage relative to bc1 monomers) by applying Equation 2. SQtotEh(%)=SQbH2+Eh(%)1−bH3+(%)1002 This equation implies that, for instance, when the bH heme is half-reduced (at Eh = 61 mV), if the EPR-detectable SQbH2+ amounts to 15%/bc1 monomer, an additional 15% of the EPR-invisible SQ is expected to be bound to center N sites in which the bH heme is oxidized. Therefore, SQ can be estimated to occupy 30% of the total center N sites, half of which have bH2+ and the other half bH3+. At a higher Eh of 120 mV, detecting 3% of SQ/bc1 monomer by EPR implies that, because only 10% of center N sites have bH2+, SQ is present in 30% of all center N sites, 90% of which have bH3+, preventing EPR detection of SQ bound to them. An important assumption made in Equation 2 is that center N binds SQ with equal affinity irrespective of the redox state of the bH hemes, which is supported by redox titrations in the bovine bc1 complex that suggest a constant Em of the bH heme in the presence or absence of SQ (7Rich P.R. Jeal A.E. Madgwick S.A. Moody A.J. Biochim. Biophys. Acta. 1990; 1018: 29-40Crossref PubMed Scopus (88) Google Scholar). As shown in Fig. 2B, the total SQ obtained using Equation 2 yielded a maximum of 40% total SQ/bc1 monomer at ∼90 mV. Because the EPR-observable SQ was zero beyond ∼150 mV, the total SQ could not be calculated at higher Eh values, as is evident from Equation 2. The Nernst equation shows that it is thermodynamically impossible to have a redox couple in which the oxidized or reduced species reaches a concentration of exactly 100%. Therefore, the denominator in Equation 2 will never reach a value of exactly zero, which would result in a mathematical indeterminacy. Also, because by definition SQbHEh2+ cannot be larger than the percentage of center N sites that have bH2+, the SQtotEh value will always be <100% of the total center N sites, even at high Eh values. For example, even at an Eh at which the bH heme is 99% oxidized, SQbH2+ will be ≤1% simply because only 1% of all the center N sites have a reduced bH heme that allows EPR detection of a bound SQ. Therefore, applying Equation 2 in this case would yield values of 1 or less in the numerator and 0.01 in the denominator, resulting in an SQtotEh value of ≤100%. The Em values of the QH2/SQ and SQ/Q couples were calculated by fitting the experimental data points SQbHEh2+ as well as the predicted SQtotEh concentrations to the following Nernst equation (Equation 3), SQEh(%)=(C)(nFeRT(Em(SQ/Q)−Eh)1+nFeRT(Em(SQ/Q)−Eh)−nFeRT(Em(QH2/SQ)−Eh)1+nFeRT(Em(QH2/SQ)−Eh))3 where C corresponds to the theoretical concentration of SQ/monomer (as a percentage) that could be achieved if the Em(SQ/Q) and Em(QH2/SQ) values were separated enough to allow maximum accumulation. Its value was ∼35% for the EPR-detectable SQ and ∼55% for the total SQ calculated from Equation 2. The fitted Eh at which maximum SQ occurred, which is the mean of the two individual Em values with their respective deviation, yielded values of 44.8 ± 7.6 mV for SQbH2+ and 86.8 ± 5.3 mV for SQtot. Therefore, the peak in total bound SQ occurred when the bH heme was 75% oxidized, implying that, at pH 7.4, SQ stabilization favors the formation of SQ·bH3+ complexes compared with SQ·bH2+ by a factor of 3. Considering that the Q pool in membranes has an Em7 reported to be between 60 (6T'sai A.L. Palmer G. Biochim. Biophys. Acta. 1983; 722: 349-363Crossref PubMed Scopus (54) Google Scholar) and 90 (9Takamiya K.I. Dutton P.L. Biochim. Biophys. Acta. 1979; 546: 1-16Crossref PubMed Scopus (99) Google Scholar, 19Rich P.R. Biochim. Biophys. Acta. 1984; 768: 53-79Crossref PubMed Scopus (315) Google Scholar) mV and that this value changes by -60 mV/pH unit, SQ bound at center N showed an Em 24–54 mV higher than unbound Q at pH 7.4. This implies that QH2 binds to center N between 2.5- and 8.3-fold tighter than Q. Reduction Kinetics of Heme bH through Center N in the Presence of DBQ—When DBH2 is added to center P-inhibited bc1 complex, electrons equilibrate with the bH hemes only by entry through center N (12Covian R. Trumpower B.L. J. Biol. Chem. 2005; 280: 22732-22740Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). As shown in Fig. 3, addition of increasing concentrations of DBQ resulted in a progressively smaller decrease in the extent of bH reduction. Interestingly, this oxidation of cytochrome b by DBQ was only partial, reaching a limit of ∼40% (with myxothiazol) or ∼60% (with stigmatellin) of the total extent observed without added DBQ. As reported previously (32Covian R. Trumpower B.L. J. Biol. Chem. 2006; 281: 30925-30932Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), bH reduction by DBH2 through center N showed biphasic kinetics when center P was inhibited with myxothiazol (Fig. 3A), whereas the presence of stigmatellin resulted in an additional re-oxidation phase that was abolished when DBQ was added (Fig. 3B). Myxothiazol and stigmatellin affected differently the way in which DBQ decreased the extent of the two kinetic components of bH reduction (Fig. 4). Only the fast phase of bH reduction was decreased in its magnitude in the presence of myxothiazol (Fig. 4A), with an estimated Km for DBQ oxidation of 12 μm. This contrasted with what was observed with stigmatellin, where the extent of reduction during the slower phase was greatly decreased by low concentrations of DBQ (Km = 1.4 μm), and the fast reduction showed a modest decrease in its extent only above 30 μm DBQ (Fig. 4B). The slower reduction phase in the presence of stigmatellin contributed much more to the total extent of reduction (45%) than that in the presence of myxothiazol (13%), although this contribution became less as DBQ was added. The rate of the fast phase of bH reduction was insensitive to DBQ concentration, irrespective of the center P inhibitor present (Fig. 5). This is a surprising result, considering that the bH oxidation effect of DBQ shown in Fig. 3 demonstrates that DBQ can bind efficiently to center N. If DBQ is assumed to bind to center N in the oxidized bc1 complex with the same affinity as that calculated from the oxidation of the bH heme, a significant decrease in the rate of reduction by DBH2 should have been expected (as simulated by the dashed lines in Fig. 5). This result indicates that DBQ does not compete with DBH2 for binding to center N when the bH heme is oxidized. DBQ decreased the rate of the slower phase of bH reduction in the presence of stigmatellin to a value similar to that observed with myxothiazol, which was unaffected by DBQ. A Km of 2.7 μm was calculated for DBQ based on this decrease in the slow rate. EPR Spectra of the Center N Semiquinone in the Presence of Center P Inhibitors—To determine the cause of the different bH reduction kinetics observed in the presence of myxothiazol and stigmatellin, we examined the properties of the EPR-detectable SQ in the absence and presence of these inhibitors (Fig. 6). The intensities and Em values of SQ in the uninhibited and myxothiazol- and stigmatellin-bound bc1 complexes were very similar, within experimental error (Fig. 6A), indicating that the center P ligand had no effect on the stability of SQ at center N. The power saturation behavior of the SQ signal was also the same under the three conditions (Fig. 6B), implying that the relaxation properties that report the electronic environment of the unpaired electron in SQ are also independent of the center P inhibitors. These results suggest that changes in center N that are responsible for differences in bH reduction kinetics when stigmatellin is present are" @default.
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