Rapid Electron Transfer between Monomers when the Cytochrome bc1 Complex Dimer Is Reduced through Center N
2005; Elsevier BV; Volume: 280; Issue: 24 Linguagem: Inglês
10.1074/jbc.m413592200
ISSN1083-351X
AutoresRaúl Covián, Bernard L. Trumpower,
Tópico(s)Porphyrin and Phthalocyanine Chemistry
ResumoWe have obtained evidence for electron transfer between cytochrome b subunits of the yeast bc1 complex dimer by analyzing pre-steady state reduction of cytochrome b in the presence of center P inhibitors. The kinetics and extent of cytochrome b reduced by quinol in the presence of variable concentrations of antimycin decreased non-linearly and could only be fitted to a model in which electrons entering through one center N can equilibrate between the two cytochrome b subunits of the bc1 complex dimer. The bH heme absorbance in a bc1 complex inhibited at center P and preincubated with substoichiometric concentrations of antimycin showed a red shift upon the addition of substrate, which indicates that electrons from the uninhibited center N in one monomer are able to reach the bH heme at the antimycin-blocked site in the other. The extent of cytochrome b reduction by variable concentrations of menaquinol could only be fitted to a kinetic model that assumes electron equilibration between center N sites in the dimer. Kinetic simulations showed that non-rate-limiting electron equilibration between the two bH hemes in the dimer through the two bL hemes is possible upon reduction through one center N despite the thermodynamically unfavorable bH to bL electron transfer step. We propose that electron transfer between cytochrome b subunits minimizes the formation of semiquinone-ferrocytochrome bH complexes at center N and favors ubiquinol oxidation at center P by increasing the amount of oxidized cytochrome b. We have obtained evidence for electron transfer between cytochrome b subunits of the yeast bc1 complex dimer by analyzing pre-steady state reduction of cytochrome b in the presence of center P inhibitors. The kinetics and extent of cytochrome b reduced by quinol in the presence of variable concentrations of antimycin decreased non-linearly and could only be fitted to a model in which electrons entering through one center N can equilibrate between the two cytochrome b subunits of the bc1 complex dimer. The bH heme absorbance in a bc1 complex inhibited at center P and preincubated with substoichiometric concentrations of antimycin showed a red shift upon the addition of substrate, which indicates that electrons from the uninhibited center N in one monomer are able to reach the bH heme at the antimycin-blocked site in the other. The extent of cytochrome b reduction by variable concentrations of menaquinol could only be fitted to a kinetic model that assumes electron equilibration between center N sites in the dimer. Kinetic simulations showed that non-rate-limiting electron equilibration between the two bH hemes in the dimer through the two bL hemes is possible upon reduction through one center N despite the thermodynamically unfavorable bH to bL electron transfer step. We propose that electron transfer between cytochrome b subunits minimizes the formation of semiquinone-ferrocytochrome bH complexes at center N and favors ubiquinol oxidation at center P by increasing the amount of oxidized cytochrome b. According to the protonmotive Q 1The abbreviations used are: Q, quinone; QH2, quinol; SQ, semiquinone; DBH2, decylubiquinol (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol); MQH2, menaquinol (2,3-dimethyl-1,4-naphthoquinol); MQ, menaquinone; MSQ, menasemiquinone; E, enzyme; I, inhibitor. cycle (1Mitchell P. J. Theor. Biol. 1976; 62: 327-367Crossref PubMed Scopus (927) Google Scholar, 2Trumpower B.L. Gennis R.B. Annu. Rev. Biochem. 1994; 63: 675-716Crossref PubMed Scopus (471) Google Scholar), the cytochrome bc1 complex is able to move charges across the membrane in which it is embedded by using one of the two electrons obtained from quinol oxidation at center P to reduce quinone bound at center N. A key feature of this cycle is that the semiquinone formed at center N after one center P turnover needs to be stabilized until the second electron from another QH2 oxidation at center P can arrive. A stable SQ at center N has been detected by EPR spectroscopy in the bc1 complex from different sources (3Siedow J.N. Power S. de la Rosa F.F. Palmer G. J. Biol. Chem. 1978; 253: 2392-2399Abstract Full Text PDF PubMed Google Scholar, 4Ohnishi T. Trumpower B.L. J. Biol. Chem. 1980; 255: 3278-3284Abstract Full Text PDF PubMed Google Scholar, 5De Vries S. Berden J.A. Slater E.C. Trumpower B.L. Function of Quinones in Energy Conserving System. Academic Press, New York1982: 235-246Google Scholar, 6Meinhardt S.W. Yang X.H. Trumpower B.L. Ohnishi T. J. Biol. Chem. 1987; 262: 8702-8706Abstract Full Text PDF PubMed Google Scholar), and this stabilization helps to explain why superoxide formation at center N is virtually non-existent when center P is blocked with stigmatellin (7Sun J. Trumpower B.L. Arch. Biochem. Biophys. 2003; 419: 198-206Crossref PubMed Scopus (136) Google Scholar, 8Muller F.L. Roberts A.G. Bowman M.K. Kramer D.M. Biochemistry. 2003; 42: 6493-6499Crossref PubMed Scopus (118) Google Scholar). However, considering that reactions at center N are fully reversible, stabilizing SQ at center N should also favor the one electron transfer from QH2 to heme bH. When heme bH is reduced, center P catalysis is slowed down, as has been observed when antimycin blocks cytochrome b reoxidation or when there is no Q available to oxidize heme bH (9Snyder C.H. Gutierrez-Cirlos E.B. Trumpower B.L. J. Biol. Chem. 2000; 275: 13535-13541Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Therefore, a mechanism should exist that avoids bH reduction through center N without destabilizing SQ at that site. Crystal structures of the bc1 complexes from various sources (10Xia 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 (876) Google Scholar, 11Zhang 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 (943) Google Scholar, 12Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 13Lange C. Hunte C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2800-2805Crossref PubMed Scopus (314) Google Scholar) show a dimeric structure in which the bL hemes of each monomer are close to each other with an edge-to-edge distance ranging from 10.4 to 11.2 Å (Fig. 1). According to electron tunneling calculations, such distance should allow electron transfer at a rate in the order of 104-105 s–1 (14Osyczka A. Moser C.C. Daldal F. Dutton P.L. Nature. 2004; 427: 607-612Crossref PubMed Scopus (222) Google Scholar). Before this structural information was available, kinetic and spectroscopic evidence was used to propose direct electron equilibration between the two center N sites of the dimer (15De Vries S. Albracht A.S.J. Berden J.A. Slater E.C. Biochim. Biophys. Acta. 1982; 681: 41-53Crossref PubMed Scopus (107) Google Scholar, 16Marres C.A. de Vries S. Biochim. Biophys. Acta. 1991; 1057: 51-63Crossref PubMed Scopus (21) Google Scholar). However, such equilibration was assumed to occur by direct electron transfer between the Q molecules bound at the two center N sites and not by electron communication between the bL hemes. Crystal structures have since shown such transfer to be impossible due to distance constraints. We have recently provided evidence that two bH hemes per bc1 dimer can be reduced through one center P when both center N sites are blocked with antimycin and that the stimulation of steady state catalysis by low concentrations of antimycin requires both monomers to use only one center N for Q reduction (17Covian 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). However, rapid mobility of an antimycin molecule between the two N sites of the dimer has also been proposed to account for non-linear inhibition of steady state activity (18Bechmann G. Weiss H. Rich P.R. Eur. J. Biochem. 1992; 208: 315-325Crossref PubMed Scopus (30) Google Scholar). In the present work, we have analyzed the pre-steady state kinetics of reduction through center N and conclude that electron equilibration between cytochrome b subunits through the bL hemes of the dimeric bc1 complex is the only model consistent with the experimental data. We also suggest that a possible function of this intermonomer electron communication is to use the stabilization of SQ at center N to maintain the bH hemes in the oxidized state, ensuring a maximal rate of QH2 oxidation at center P. Materials—Dodecylmaltoside was obtained from Roche Applied Science. DEAE-Bio-Gel was obtained from Bio-Rad Laboratories. Stigmatellin was from Fluka. Antimycin, myxothiazol, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, horse heart cytochrome c, decylubiquinone, sodium ascorbate, sodium dithionite, and sodium borohydride were purchased from Sigma. MQ was synthesized in the laboratory. DBH2 and MQH2 were prepared as described before (19Trumpower B.L. Edwards C.A. J. Biol. Chem. 1979; 254: 8697-8706Abstract Full Text PDF PubMed Google Scholar, 20Snyder 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 (21Gutierrez-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) by using reported extinction coefficients (22Von Jagow G. Link T.A. Methods Enzymol. 1986; 126: 253-271Crossref PubMed Scopus (315) Google Scholar, 23Rich P.R. Biochim. Biophys. Acta. 1984; 768: 53-79Crossref PubMed Scopus (317) Google Scholar). MQH2 was quantified by determining the amount of cytochrome c reduced by 50 nmbc1 complex, assuming two cytochrome c molecules reduced per MQH2 oxidized. Purification of Cytochrome bc1 Complex—Cytochrome bc1 complex was isolated from Red Star cake yeast as described previously (19Trumpower B.L. Edwards C.A. J. Biol. Chem. 1979; 254: 8697-8706Abstract Full Text PDF PubMed Google Scholar, 24Ljungdahl P.O. Pennoyer J.D. Robertson D.E. Trumpower B.L. Biochim. Biophys. Acta. 1987; 891: 227-241Crossref PubMed Scopus (127) Google Scholar). Quantification of the bc1 complex was performed as reported before (20Snyder C.H. Trumpower B.L. Biochim. Biophys. Acta. 1998; 1365: 125-134Crossref PubMed Scopus (44) Google Scholar) by using extinction coefficients of 17.5 mm–1 cm–1 at 553–539 for cytochrome c1 (25Yu 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 563–579 for the average absorbance of the bH and bL heme in cytochrome b (26Berden J.A. Slater E.C. Biochim. Biophys. Acta. 1970; 216: 237-249Crossref PubMed Scopus (152) Google Scholar). Quantification of Endogenous Ubiquinone in Purified bc1 Complex— A modification of the procedure reported by Kröger (27Kröger A. Methods Enzymol. 1978; 53: 579-591Crossref PubMed Scopus (121) Google Scholar) was used. Briefly, 100 μlofa30–80 μm solution of purified bc1 complex was mixed with 2.4 ml of a mixture of 60% methanol: 40% cyclohexane (v/v) in a 10-ml glass tube with screw cap. The upper phase containing cyclohexane was collected, and the lower layer was re-extracted with 1 ml of cyclohexane. The cyclohexane phases were evaporated under an argon gas flow, and the yellowish residue was dissolved in 1 ml of ethanol. UV spectra of these samples were recorded in the range of 240–340 nm by using an Aminco DW-2 dual wavelength spectrophotometer. Spectra were recorded before and after the addition of 5 μl of 0.5 m sodium borohydride. The reduced spectra were subtracted from the oxidized spectra, and Q content was calculated from these difference spectra by using an extinction coefficient of 12.9 mm–1cm–1 at 275 nm. The average content of endogenous Q in three different purified bc1 complex preparations was found to be 1.08 ± 0.08 Q/monomer. Pre-steady State Reduction of bc1 Complex—Pre-steady state reduction of cytochrome b was followed at room temperature by stopped flow rapid scanning spectroscopy using the OLIS rapid scanning monochromator (20Snyder 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 assay buffer containing 50 mm phosphate, pH 7.0, 250 mm sucrose, 1 mm sodium azide, 0.2 mm EDTA, 0.05% Tween 20, 6 μm stigmatellin or myxothiazol, and variable concentrations of antimycin (between 0 and 3 μm) against an equal volume of the same buffer, without enzyme and inhibitors, containing different concentrations of DBH2 or MQH2. For each experiment, 6–8 data sets were averaged, and the oxidized spectrum was subtracted. The time course of absorbance change at 563 and 578 nm was extracted by using software from OLIS. Kinetic Modeling—The Dynafit program (Biokin, Ltd.) used for kinetic modeling 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 (28Kuzmic P. Anal. Biochem. 1996; 237: 260-273Crossref PubMed Scopus (1360) Google Scholar). The mechanism is described as a series of individual reaction steps. A family of kinetic traces in which 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. In the models used in the present work, extinction coefficients of 36 and 15 mm–1cm–1 were used for hemes bH and bL, respectively. These values come from the reported extinction coefficient of 51.2 mm–1cm–1 for a fully reduced cytochrome b monomer (usually expressed as 25.6 mm–1cm–1 for the average absorbance of both b hemes), taking into consideration that bH contributes ∼70% of this value (26Berden J.A. Slater E.C. Biochim. Biophys. Acta. 1970; 216: 237-249Crossref PubMed Scopus (152) Google Scholar, 29Rich P.R. Jeal A.E. Madgwick S.A. Moody A.J. Biochim. Biophys. Acta. 1990; 1018: 29-40Crossref PubMed Scopus (88) Google Scholar). Since the mixing chamber in the OLIS-rapid scanning monochromator has a path length of 2 cm, full bH reduction of the bc1 complex present in the reaction (1.5 μm) would result in a Δ absorbance value at 563–579 of ∼0.108, whereas full bH and bL reduction should yield Δ absorbance = 0.154. Dynafit also allows modeling of an equilibration phase in which two or more ligands interact before dilution and the addition of other reactants. A simplified explanation of the models used in fitting and simulating data is provided below. The complete script files are provided as Supplemental Data. For fitting of pre-steady state kinetics of cytochrome b reduction by QH2 through center N in the presence of variable antimycin concentrations, two models were used. One of the models assumed that the dimeric oxidized enzyme (E) bound tightly one or two inhibitor (I) molecules in a random, non-cooperative manner before the addition of substrate to form three possible complexes (E.I, I.E, and I.E.I). Then, binding of QH2 was allowed to form five possible productive complexes (E.QH2,QH2.E, QH2.E.QH2, I.E.QH2, and QH2.E.I). Electron transfer reactions were assumed to occur for each of these complexes as shown in Fig. 2, Scheme 1, in which reactions for only two of all possible complexes (I.E.QH2 and QH2.E.I) are depicted. This model assumed that an electron in the bH heme of one monomer (EbH or bHE) could be transferred to the bH heme in the other monomer reversibly in a single step (with k3 = k-3), which would include intermediate reactions occurring through the bL hemes not explicitly considered in this particular model. The result of this intradimer equilibration is that both bH hemes can be reduced through only one center N in the dimer. This is illustrated in Scheme 1, in which both bH hemes in QH2.E.I and I.E.QH2 can be reduced to yield Q.bHEbH.I and I.bHEbH.Q. In this model, SQ was considered to be a tightly bound ligand, not dissociating from E. The second model omitted the intermonomer electron transfer step and included instead the movement of I from one monomer to the other (18Bechmann G. Weiss H. Rich P.R. Eur. J. Biochem. 1992; 208: 315-325Crossref PubMed Scopus (30) Google Scholar), as shown in Fig. 2, Scheme 2. In this model, QH2 (Em = 90 mV) is not able to reduce heme bL (Em = –30 mV, see Ref. 30Tsai A.L. Palmer G. Biochim. Biophys. Acta. 1983; 722: 349-363Crossref PubMed Scopus (54) Google Scholar) significantly to form Q, which would then be able to leave center N. Thus, SQ had to be assumed to dissociate, allowing the inhibitor to move from the oxidized to the reduced bH monomer. This made it possible for QH2 to bind and reduce the second bH in the dimer, as illustrated in Fig. 2, Scheme 2, in which I.bHEbH.SQ and SQ.bHEbH.I can only be formed after SQ dissociates from SQ.bHE.I and I.EbH.SQ. This dissociation was considered to be irreversible due to the reactivity of the highly reducing SQ molecule outside center N. Movement of I between monomers was described by a rate constant (kim) different from the dissociation rate constant that would result in the formation of free I (kdI). Reduction of cytochrome b by MQH2 through center N in the absence of antimycin was also fitted to two different models. One of them was a modified version of the mechanism explained in Fig. 2, Scheme 1 in which equilibration of one molecule of endogenous Q per bc1 complex monomer was allowed before dilution and mixing with MQH2. For simplicity, no distinction was made between the monomers, as was done in the models described above. Electron transfer between monomers was implicitly included by making ligands in either site exchange electrons with either one of the bH hemes. This is illustrated in Fig. 2, Scheme 3, in which some of the possible reactions included for E.MQH2.Q are shown. The same sequence of possible reactions would apply for E.MQH2 and E.MQH2.MQH2, with additional binding and dissociation of ligands from any of the two center N sites of the dimer. Only the semiquinones SQ and MSQ were assumed not to dissociate from the enzyme. Reduction of bL was included in additional reaction steps from EbHbH complexes. To reduce script size, binding of MQ and QH2 were not written as separate reactions but were instead included in the corresponding electron transfer step in which they participated. This is reasonable considering that the reactions involving these two ligands will be mostly unidirectional, with MQ (Em = –70 mV) rarely taking electrons from bH (Em = 120 mV) and QH2 being formed in low concentrations from the two-electron reduction of only 1 eq of endogenous Q per monomer. The second model used considered E to be only a monomeric bc1 complex, with no possibility of intermonomer electron transfer. This linear model describes a transhydrogenation reaction in which electrons from MQH2 can only be transferred to Q after both bH and bL hemes in a monomer have been reduced so that MQ leaves center N and Q binds to oxidize the fully reduced cytochrome b. This mechanism is illustrated in Fig. 2, Scheme 4. The only rate constants that were fixed during the fitting procedure were kb and k–b, which describe the electron transfer between the bL and bH hemes. Considering that equilibration between both hemes occurs within a few μs, the rate from bL to bH (kb) was set to 105 s–1. To fix the value for the reverse rate constant from bH to bL (k–b), the following thermodynamic considerations were made. The equilibrium constant (Keq) for electron transfer between both hemes can be calculated from the Nernst equation at 25 °C. ΔEm=59.2mVlog[oxbH][redbL][redbH][oxbL](Eq. 1) Keq=[oxbH][redbL][redbH][oxbL]=10ΔEm59.2mV(Eq. 2) Using a difference in redox potential (ΔEm) between the two b hemes in yeast of –150 mV (30Tsai A.L. Palmer G. Biochim. Biophys. Acta. 1983; 722: 349-363Crossref PubMed Scopus (54) Google Scholar), an equilibrium constant (Keq) of 0.0029 for the electron transfer from bH to bL is obtained. However, to calculate the ratio of [redbL]/[redbH] when only one electron is moving between the two hemes, the following applies [oxbH]=[redbL][redbH]=[oxbL](Eq. 3) As a result of these assumptions, the Nernst equation can be expressed as ΔEm=59.2mVlog[redbL]2[redbH]2(Eq. 4) Rearranging, we obtain [redbL][redbH]=10ΔEm2(59.2mV)(Eq. 5) For ΔEm = –150 mV, the [redbL]/[redbH] ratio when one electron is equilibrating within a cytochrome b monomer is 0.054. Therefore, a ratio of k–b/kb = 0.054 was also used for those reactions in Fig. 2, Scheme 4 describing electron transfer between bH and bL. Since kb was chosen to be 105 s–1, k–b was set to 5400 s–1. It is noteworthy that the ratio of these unimolecular rate constants is not the same as the value obtained for the formal Keq from Equation 2 (0.0029) but is equal to Keq. This comes from the fact that the kb/k–b ratio was used to express the [redbL]/[redbH] ratio as deduced from Equations 3, 4, 5 and not the complete ratio of [oxbH][redbL]/[redbH][oxbL], which would require bimolecular constants (in units of M–1s–1) to be used. Determination of the Antimycin-induced Red Shift of Heme bH Absorbance—Red shifts induced by antimycin upon reduction with substrate after inhibitor addition were determined after incubating 3 μm yeast enzyme for at least 2 min with 6 μm stigmatellin and different concentrations of antimycin in assay buffer before rapid mixing against an equal volume of buffer containing 24 μm MQH2 by using the OLIS rapid scanning monochromator. The kinetic traces showed no further change in cytochrome b absorbance measured at 568–579 nm after 1.2 s of collection. Therefore, the 800 spectra collected between 1.2 and 2.0 s were averaged by using the OLIS software. The absorbance values for all spectra were exported and analyzed with the Origin 5.0 program (OriginLab Corp.). The averaged spectrum without antimycin cannot be used directly as a reference for the spectra with antimycin because of its larger absorbance. Therefore, a normalization procedure was followed to generate a calculated reference spectrum for each of the spectra with antimycin. First, the absorbance of all spectra was made equal to zero at the reference wavelengths for cytochrome b (579 nm) by subtracting the absorbance of all wavelengths from the value at 579 nm. Then, the absorbance of each of the spectra at the reference wavelength for cytochrome c1 (539 nm) was also made equal to zero while maintaining the absorbance value at 579 nm fixed, applying the following equation. Absλf=Absλi−(579−λ579−539)Abs539(Eq. 6) Absλf is the final absorbance value at a particular wavelength (λ), and Absλi is the initial absorbance at that same wavelength. This equation in effect changes the slope of an imaginary line between 539 and 579 nm to a value of zero. Therefore, the final result is that all spectra will have zero absorbance at both wavelengths while preserving the shape of the spectra, similar to a baseline correction. The final step involves normalizing the absorbance of the spectrum taken with no antimycin to each of the other spectra separately. Since the maximum absorbance of the spectrum without antimycin was at 563 nm, this wavelength was used for the normalization according to the following equation. AbsλfnI0=AbsλiI0(Abs563InAbs563iI0)(Eq. 7) AbsλfnI0 corresponds to the final absorbance at each wavelength of the spectrum with zero antimycin that is used as reference for the spectrum taken with n equivalents of antimycin per bc1 complex monomer. This generated a unique reference spectrum for each of the spectra with inhibitor. AbsλfnI0 is the initial absorbance at each wavelength of the spectrum with no antimycin (after applying Equation 6 above). Abs563In is the absorbanceIat 563 nm of the spectrum taken with n equivalents of antimycin. Abs563iI0 corresponds to the initial absorbance at 563 nm of the spectrum with no antimycin that resulted from applying Equation 6. For example, if the absorbance at 563 Inm of a spectrum taken with a certain concentration of antimycin (Abs563In) was 0.03, and the absorbance at that same wavelength in the spectrum with no antimycin (Abs563iI0) was 0.06, the absorbance at all wavelengths of the spectrum with no antimycin (AbsλiI0) would be multiplied by 0.5 to obtain the reference spectrum. The normalized absorbance at each wavelength this reference spectrum of (AbsλfnI0) was then subtracted from the absorbance at each wavelength Iof the spectrum with that particular concentration of antimycin (AbsλIn), as expressed in Equation 8. ΔAbsλIn=AbsλIn−AbsλfnI0(Eq. 8) The difference spectra for each n antimycin concentration (ΔAbsλIn) had zero absorbance at 539, 563, and 579 nm because of the normalization procedure described above. This resulted in red shift spectra with larger absolute absorbance values for the peak than for the trough. However, the absolute absorbance difference between peak and trough is independent of this asymmetry, and therefore, the magnitude of the red shift can still be taken as a direct measure of antimycin bound to a center N, where the bH heme underwent reduction after the addition of the inhibitor. Titration of the Pre-steady State Reduction of Cytochrome b through Center N with Antimycin—When center P was blocked, increasing concentrations of antimycin at intervals of 0.1 mol/mol of bc1 monomer modified the kinetics of cytochrome b reduction by DBH2 as shown in Fig. 3. A good fit to the experimental data (Fig. 3A) was obtained by using a model that allowed electrons to move between monomers as described in Fig. 2, Scheme 1, and under "Experimental Procedures." A fitted value of 21.1 s–1 was obtained for the intradimer electron transfer rate (k3 in Fig. 2, Scheme 1, see Supplemental Data for fitted values of all other rate constants). In contrast, fitting to a model assuming fast antimycin movement between monomers and no intradimer electron transfer yielded sharply biphasic kinetics and a larger extent of reduction than experimentally observed, especially at low antimycin concentrations (Fig. 3B). This was a consequence of the SQ dissociation step (fitted value of k1 dSQ = 3.4 s–) included in the model, which was needed to allow movement of an inhibitor molecule from an oxidized to a reduced monomer (Fig. 2, Scheme 2). According to this model, the excess of QH2 present (16 mol of DBH2/monomer) should result in full bH reduction in both monomers (ΔAbs ∼0.1) when little or no inhibitor is present, which was clearly not the case. Furthermore, the fitted value for the rate of intermonomer movement of antimycin (kim = 1.9 × 109 s–1) had a standard error several orders of magnitude higher than the fitted value. This means that any value for this constant could be used with little effect on the overall kinetics because movement of antimycin from one monomer to the other would be limited by the rate of SQ dissociation (kdSQ). Modifying the model by allowing SQ dissociation to be reversible by including an association rate constant (kaSQ) with a separate irreversible step describing SQ autoxidation to Q outside center N did not improve the fitting (data not shown). When the total extent of cytochrome b reduction was plotted as a function of antimycin concentration, non-linear titration curves were obtained (Fig. 4). This occurred independently of the substrate (DBH2 or MQH2) or center P inhibitor (stigmatellin or myxothiazol) used. Titrating center N with another inhibitor such as ilicicolin (31Gutierrez-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) yielded the same results (not shown). These non-linear titration curves could be fitted by assuming that the same extent of reduction could be obtained when either one or both center N sites per dimer were not bound by antimycin (Fig. 4, solid lines). This implies that both bH hemes in a dimer can equilibrate with the substrate through only one center N, as assumed in the model used for fitting in Fig. 3A. In contrast, if each bH heme is assumed to be reducible only through its adjacent center N in the same monomer, linear inhibition would be expected (Fig. 4, dashed lines). Linear binding of antimycin to center N was observed by measuring the red shift induced in the spectrum of dithionite-reduced bH upon binding of the inhibitor (not shown), as has been reported in other bc1 complexes (32Van den Berg W.H. Prince R.C. Bashford L. Takamiya K. Bonner W.D. Dutton P.L. J. Biol. Chem. 1979; 254: 8594-8604Abstract Full Text PDF PubMed Google Scholar). This eliminates the possibility that non-linearity in the extent of cytochrome b reduction was due to poor accessibility of antimycin to center N at low inhibitor concentrations due to the presence of detergent. Reduction of bH Heme in the Antimycin-blocked Center N— Hydrophobic tightly bound inhibitors similar to antimycin dissociate very slowly from their binding sites, with dissociation rates in the order of at least minutes (33Zhang L. Snyder C. Trumpower B.L. Yu L. Yu C.A. FEBS Lett. 1999; 460: 349-352Crossref PubMed Scopus (9) Google Scholar, 34Covian R. Pardo J.P. Moreno-Sanchez R. J. Biol. Chem. 2002; 277: 48449-48455Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). This means that antimycin bound at a particular center N will not dissociate within the 1–2 s needed to reduce cytochrome b with QH2 or MQH2, thus preventing bH reduction at that cente
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