Triphasic reduction of cytochrome b and the protonmotive Q cycle pathway of electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain

Reduction of cytochrome b in isolated succinate-cytochrome c reductase is a triphasic reaction. Initially, there is a relatively rapid, partial reduction of the cytochrome b, the rate of which matches the rate of reduction of cytochrome c1. This is followed by partial or complete reoxidation of the b, which is then followed by slow rereduction. At very low concentrations of succinate, the initial partial reduction of b is followed by reoxidation, but the third (rereduction) phase is absent, owing to insufficient substrate to rereduce the cytochromes. If antimycin is added at various times during the triphasic reaction, it inhibits the reoxidation and also inhibits the rereduction phase. Antimycin does not inhibit the initial phase of b reduction and, if added before or during this phase, it causes reduction of b to proceed to completion as a monophasic reaction. Myxothiazol inhibits the first phase of b reduction and the subsequent reoxidation, but does not inhibit the third, slow phase of b reduction. The resulting monophasic reduction of b which is observed in the presence of myxothiazol is slower than that in the presence of antimycin. The combination of both inhibitors, whether added together or successively during the triphasic reaction, completely inhibits b reduction. The triphasic reduction of cytochrome b is consistent with electron transfer by a protonmotive Q cycle in which there are two pathways for cytochrome b reduction. One pathway allows the initial phase of cytochrome b reduction by a myxothiazol-sensitive reaction in which reduction of b by ubisemiquinone is linked to reduction of iron-sulfur protein and cytochrome c1 by ubiquinol. In the second phase of the triphasic reaction, the b cytochromes are reoxidized by ubiquinone or ubisemiquinone through an antimycin-sensitive reaction. If oxidation of ubiquinol by iron-sulfur protein is blocked, either by myxothiazol or by reduction of iron-sulfur protein and cytochrome c1, the b cytochromes can be reduced by reversal of the antimycin-sensitive pathway, thus accounting for the third phase of b reduction.     

The interaction of yeast Complex III with some respiratory inhibitors

We have examined the effects of eight inhibitors of the bovine-heart mitochondrial Complex III on the catalytic activity of the analogous complex from yeast mitochondria. All eight compounds were inhibitory, with potent inhibition being obtained with antimycin, myxothiazol and UHDBT (5-N-undecyl-6-hydroxy-4,7-dioxobenzothiazole). These three inhibitors, and also funiculosin, have been further studied by characterizing their effects on the visible absorbance, magnetic circular dichroism and EPR spectra of the complex and also on the potentiometric properties of the individual metal centers present in the complex. All four inhibitors had little or no effect on either the absorbance or magnetic circular dichroism spectra. Funiculosin produced a change in the EPR lineshape of the iron-sulfur cluster; EPR spectra recorded at 12 K also revealed complete reduction of cytochrome b-562 by ascorbate. UHDBT also changed the lineshape of the iron-sulfur cluster and this change could be partially reversed by myxothiazol. Neither antimycin nor myxothiazol affected the iron-sulfur cluster and produced only small changes in the EPR absorption envelope of the b cytochromes. Both funiculosin and UHDBT raised the midpoint potential of the iron-sulfur cluster, by about 150 and 70 mV, respectively. Only UHDBT changed the potential of c1, lowering it by about 30 mV. Funiculosin raised the potential of b-562 by about 30 mV, while myxothiazol had no effect; the other two compounds produced only small changes. All four compounds had only small effects on the midpoint potential of b-566. The relative contributions of the two b cytochromes to the magnetic circular dichroism amplitudes could be changed by the addition of inhibitors, even though the absolute magnetic circular dichroism spectra of oxidized and reduced complex were unaffected.     

The kinetics of reduction of yeast complex III by a substrate analog

The kinetics of reduction of the cytochrome and quinone constituents of yeast complex III by the substrate homolog Q1H2 have been measured under a variety of conditions. The maximum rates of reduction of cytochromes b and c1 and of the endogenous Q6 by Q1H2 were sufficiently fast to support the Vmax for the reduction of cytochrome c by this substrate. The absorbance at 562 nm showed an initial increase which was subsequently followed by a decrease. This decrease was synchronous with the appearance of reduced cytochrome c1 and is interpreted as reflecting the absorbance contribution of c1 at 562 nm under conditions where the steady state level of the b cytochromes is constant. Prereduction of c1 and the Fe/S cluster did not affect the initial very rapid reduction of b, but the second phase was eliminated. Antimycin abolished the very rapid rate of reduction of cytochrome b in untreated complex III and completely inhibited the reduction of cytochrome b in complex III in which c1 and the Fe/S cluster had been prereduced. However, the reduction of the endogenous quinone was essentially unaffected by these treatments. Antimycin had no effect on the reduction of c1. Funiculosin also suppressed the very rapid reduction of b while both myxothiazol and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole did not modify this phase of the reaction; no secondary decrease in absorbance was observed in the presence of any of these inhibitors. Most of the observed kinetic changes could be reproduced by simulation of the Q-cycle; simple linear and branched schemes were unable to reproduce the data.     

Ubiquinol:cytochrome c oxidoreductase (complex III). Effect of inhibitors on cytochrome b reduction in submitochondrial particles and the role of ubiquinone in complex III

Two sets of studies have been reported on the electron transfer pathway of complex III in bovine heart submitochondrial particles (SMP). 1) In the presence of myxothiazol, MOA-stilbene, stigmatellin, or of antimycin added to SMP pretreated with ascorbate and KCN to reduce the high potential components (iron-sulfur protein (ISP) and cytochrome c(1)) of complex III, addition of succinate reduced heme b(H) followed by a slow and partial reduction of heme b(L). Similar results were obtained when SMP were treated only with KCN or NaN(3), reagents that inhibit cytochrome oxidase, not complex III. The average initial rate of b(H) reduction under these conditions was about 25-30% of the rate of b reduction by succinate in antimycin-treated SMP, where both b(H) and b(L) were concomitantly reduced. These results have been discussed in relation to the Q-cycle hypothesis and the effect of the redox state of ISP/c(1) on cytochrome b reduction by succinate. 2) Reverse electron transfer from ISP reduced with ascorbate plus phenazine methosulfate to cytochrome b was studied in SMP, ubiquinone (Q)-depleted SMP containing 相似文献   

The oxidation of yeast Complex III. Evidence for a very rapid electron equilibration between cytochrome c1 and the iron-sulfur center

The reoxidation of reduced cytochrome c1 by potassium ferricyanide follows pseudo-first order kinetics with k = 4 x 10(4) M-1 s-1. However, the reoxidation of this cytochrome in two-electron reduced Complex III does not follow any simple rate law although the overall rate of reaction is essentially unchanged. The observed kinetics can be well fitted with a model in which ferricyanide reacts exclusively with cytochrome c1 together with very rapid electron transfer from the reduced iron-sulfur center to cytochrome c1. Neither removal of coenzyme Q from the complex nor prior incubation with antimycin A had any effect on the observed kinetics of reoxidation.     

Function of the iron-sulfur protein of the cytochrome b-c1 segment in electron transfer reactions of the mitochondrial respiratory chain

Resolution and reconstitution has been used to examine the involvement of the iron-sulfur protein of the cytochrome b-c1 segment in electron transfer reactions in this region of the mitochondrial respiratory chain. The iron-sulfur protein is required for electron transfer from succinate and from ubiquinol to cytochrome c1. It is not required for reduction of cytochrome b under these conditions, but it is required for oxidation of cytochrome b by cytochrome c plus cytochrome c oxidase. Removal of the iron-sulfur protein from the b-c1 complex prevents reduction of both cytochromes b and c1 by succinate or ubiquinol if antimycin is added to the depleted complex. As increasing amounts of iron-sulfur protein are reconstituted to the depleted complex, the amounts of cytochromes b and c1 reduced by succinate in the presence of antimycin increase and closely parallel the amounts of ubiquinol-cytochrome c reductase activity restored to the reconstituted complex, measured before addition of antimycin. The function of the iron-sulfur protein in these oxidation-reduction reactions is consistent with a cyclic pathway of electron transfer through the cytochrome b-c1 complex, in which the iron-sulfur protein functions as a ubiquinol-cytochrome c1/ubisemiquinone-cytochrome b oxidoreductase.     

The electric field generated by photosynthetic reaction center induces rapid reversed electron transfer in the bc1 complex

The cytochrome bc(1) complex is the central enzyme of respiratory and photosynthetic electron-transfer chains. It couples the redox work of quinol oxidation and cytochrome reduction to the generation of a proton gradient needed for ATP synthesis. When the quinone processing Q(i)- and Q(o)-sites of the complex are inhibited by both antimycin and myxothiazol, the flash-induced kinetics of the b-heme chain, which transfers electrons between these sites, are also expected to be inhibited. However, we have observed in Rhodobacter sphaeroides chromatophores, that when a fraction of heme b(H) is reduced, flash excitation induces fast (half-time approximately 0.1 ms) oxidation of heme b(H), even in the presence of antimycin and myxothiazol. The sensitivity of this oxidation to ionophores and uncouplers, and the absence of any delay in the onset of this reaction, indicates that it is due to a reversal of electron transfer between b(L) and b(H) hemes, driven by the electrical field generated by the photosynthetic reaction center. In the presence of antimycin A, but absence of myxothiazol, the second and following flashes induce a similar ( approximately 0.1 ms) transient oxidation of approximately 10% of the cytochrome b(H) reduced on the first flash. From the observed amplitude of the field-induced oxidation of heme b(H), we estimate that the equilibrium constant for sharing one electron between hemes b(L) and b(H) is 10-15 at pH 7. The small value of this equilibrium constant modifies our understanding of the thermodynamics of the Q-cycle, especially in the context of a dimeric structure of bc(1) complex.     

Ubiquinol-cytochrome c oxidoreductase of higher plants. Isolation and characterization of the bc1 complex from potato tuber mitochondria.

A procedure is described for isolation of active ubiquinol-cytochrome c oxidoreductase (bc1 complex) from potato tuber mitochondria using dodecyl maltoside extraction and ion exchange chromatography. The same procedure works well with mitochondria from red beet and sweet potato. The potato complex has at least 10 subunits resolvable by gel electrophoresis in the presence of dodecyl sulfate. The fifth subunit carries covalently bound heme. The two largest ("core") subunits either show heterogeneity or include a third subunit. The purified complex contains about 4 mumol of cytochrome c1, 8 mumol of cytochrome b, and 20 mumol of iron/g of protein. The complex is highly delipidated, with 1-6 mol of phospholipid and about 0.2 mol of ubiquinone/mol of cytochrome c1. Nonetheless it catalyzes electron transfer from a short chain ubiquinol analog to equine cytochrome c with a turnover number of 50-170 mol of cytochrome c reduced per mol of cytochrome c1 per s, as compared with approximately 220 in whole mitochondria. The enzymatic activity is stable for weeks at 4 degrees C in phosphate buffer and for months at -20 degrees C in 50% glycerol. The activity is inhibited by antimycin, myxothiazol, and funiculosin. The complex is more resistant to funiculosin and diuron than the beef heart enzyme. The optical difference spectra of the cytochromes were resolved by analysis of full-spectrum redox titrations. The alpha-band absorption maxima are 552 nm (cytochrome c1), 560 nm (cytochrome b-560), and 557.5 + 565.5 nm (cytochrome b-566, which has a split alpha-band). Extinction coefficients appropriate for the potato cytochromes are estimated. Despite the low lipid and ubiquinone content of the purified complex, the midpoint potentials of the cytochromes (257, 51, and -77 mV for cytochromes c1, b-560, and b-566, respectively) are not very different from values reported for whole mitochondria. EPR spectroscopy shows the presence of a Rieske-type iron sulfur center, and the absence of centers associated with succinate and NADH dehydrogenases. The complex shows characteristics associated with a Q-cycle mechanism of redox-driven proton translocation, including two pathways for reduction of b cytochromes by quinols and oxidant-induced reduction of b cytochromes in the presence of antimycin.     

Ubiquinol:cytochrome c oxidoreductase. Effects of inhibitors on reverse electron transfer from the iron-sulfur protein to cytochrome b

The effects of inhibitors on the reduction of the bis-heme cytochrome b of ubiquinol: cytochrome c oxidoreductase (complex III, bc1 complex) has been studied in bovine heart submitochondrial particles (SMP) when cytochrome b was reduced by NADH and succinate via the ubiquinone (Q) pool or by ascorbate plus N,N,N', N'-tetramethyl-p-phenylenediamine via cytochrome c1 and the iron-sulfur protein of complex III (ISP). The inhibitors used were antimycin (an N-side inhibitor), beta-methoxyacrylate derivatives, stigmatellin (P-side inhibitors), and ethoxyformic anhydride, which modifies essential histidyl residues in ISP. In agreement with our previous findings, the following results were obtained: (i) When ISP/cytochrome c1 were prereduced or SMP were treated with a P-side inhibitor, the high potential heme bH was fully and rapidly reduced by NADH or succinate, whereas the low potential heme bL was only partially reduced. (ii) Reverse electron transfer from ISP/c1 to cytochrome b was inhibited more by antimycin than by the P-side inhibitors. This reverse electron transfer was unaffected when, instead of normal SMP, Q-extracted SMP containing 200-fold less Q (0. 06 mol Q/mol cytochrome b or c1) were used. (iii) The cytochrome b reduced by reverse electron transfer through the leak of a P-side inhibitor was rapidly oxidized upon subsequent addition of antimycin. This antimycin-induced reoxidation did not happen when Q-extracted SMP were used. The implications of these results on the path of electrons in complex III, on oxidant-induced extra cytochrome b reduction, and on the inhibition of forward electron transfer to cytochrome b by a P-side plus an N-side inhibitor have been discussed.     

Reduction of cytochrome b in mitochondria from yeast lacking coenzyme Q

Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase but had comparable amounts of cytochromes b and c1 as wild-type mitochondria. Addition of succinate to the mutant mitochondria resulted in a slight reduction of cytochrome b; however, the subsequent addition of antimycin resulted in a biphasic reduction of cytochrome b, leading to reduction of 68% of the total dithionite-reducible cytochrome b. No "red" shift in the absorption maximum was observed, and no cytochrome c1 was reduced. The addition of either myxothiazol or alkylhydroxynaphthoquinone blocked the reduction of cytochrome b observed with succinate and antimycin, suggesting that the reduction of cytochrome b-562 in the mitochondria lacking coenzyme Q may proceed by a pathway involving cytochrome b at center o where these inhibitors block. Cyanide did not prevent the reduction of cytochrome b by succinate and antimycin the the mutant mitochondria. These results suggest that the succinate dehydrogenase complex can transfer electrons directly to cytochrome b in the absence of coenzyme Q in a reaction that is enhanced by antimycin. Reduced dichlorophenolindophenol (DCIP) acted as an effective bypass of the antimycin block in complex III, resulting in oxygen uptake with succinate in antimycin-treated mitochondria. By contrast, reduced DCIP did not restore oxygen uptake in the mutant mitochondria, suggesting that coenzyme Q is necessary for the bypass. The addition of low concentrations of DCIP to both wild-type and mutant mitochondria reduced with succinate in the presence of antimycin resulted in a rapid oxidation of cytochrome b perhaps by the pathway involving center o, which does not require coenzyme Q.     

Dynamic control on the rate of the reduction of the b type cytochromes in submitochondrial particles.

1. In the presence of antimycin and KCN the reduction of cytochrome b in phosphorylating submitochondrial particles followed a biphasic first-order kinetics. The transition from the first, rapid phase to the second, slow phase occurred while the reduction of chtochromes c + c1 and a through or around the antimycin block was still linear with time. Thus, the phase transition was due to a fall-off in the rate of cytochrome b reduction. 2. The biphasic reduction of cytochrome b was observed over a wide temperature range (0--30 degrees C), with succinate of NADH as electron donors and with phosphorylating particles or coupled rat-heart mitochondria. With rat-heart mitochondria the same biphasic reduction was observed in the presence of either carbonyl cyanide p-trifluoromethoxyphenylhydrazone or oligomycin. 3. In both the rapid and the slow phases, the rate of reduction of cytochrome b-561 was equal to that of b-565. Thus both cytochromes b-561 and b-565 were affected by the mechanism which determined the reduction-rate. Furthermore, each of these cytochromes could be reduced individually with rate constants typical of the slow phase. 4. The proportion of rapidly reduced to slowly reduced cytochrome b was independent of the degree of its reducibility and could be controlled by teh experimental conditions. When antimycin was used as the only inhibitor, 96% of the b-type cytochromes were reduced in the rapid phase. If the c and a-type cytochromes were first reduced by ascorbate and tetramethyl-p-phenylenediamine in the presence of KCN and antimycin, all the b-type cytochromes were fully reduced at the slow-rate. 5. With succinate, the rate of the rapid phase depended on the activation level of the succinic-dehydrogenase. The rate constant of the second phase was unaffected by the succinic dehydrogenase activity, if the preparation was more than 20% active. Furthermore, the rate constant of the slow reduction was the same with succinate, NADH, or even with durohydroquinone (which reacted directly with cytochromes b). 6. It is suggested that cytochrome b can exist in two forms: kinetically active or sluggish. The active form is rapidly reduced by the endogenous quinone (QH2) or durohydroquinone. The rate of the reduction of the active form by succinate or NADH is probably determined by the rate of the reduction of Q by the dehydrogenases. The second form of cytochrome b is characterized by its sluggish reduction by QH2 or durohydroquinone. 7. It is proposed that the transformation from the active to the sluggish form is induced by the reduction of a controlling group, named Y, located on the oxygen side of the antimycin inhibition site. When Y is oxidized, cytochrome b is in its active form, and when Y is reduced, cytochrome b is in its sluggish form. The nature of this kinetic control and a comparison with the mechanism controlling the reducibility of cytochrome b are discussed.     

Evidence for a concerted mechanism of ubiquinol oxidation by the cytochrome bc1 complex

To better understand the mechanism of divergent electron transfer from ubiquinol to the iron-sulfur protein and cytochrome b(L) within the cytochrome bc(1) complex, we have examined the effects of antimycin on the presteady state reduction kinetics of the bc(1) complex in the presence or absence of endogenous ubiquinone. When ubiquinone is present, antimycin slows the rate of cytochrome c(1) reduction by approximately 10-fold but had no effect upon the rate of cytochrome c(1) reduction in bc(1) complex lacking endogenous ubiquinone. In the absence of endogenous ubiquinone cytochrome c(1), reduction was slower than when ubiquinone was present and was similar to that in the presence of ubiquinone plus antimycin. These results indicate that the low potential redox components, cytochrome b(H) and b(L), exert negative control on the rate of reduction of cytochrome c(1) and the Rieske iron-sulfur protein at center P. If electrons cannot equilibrate from cytochrome b(H) and b(L) to ubiquinone, partial reduction of the low potential components slows reduction of the high potential components. We also examined the effects of decreasing the midpoint potential of the iron-sulfur protein on the rates of cytochrome b reduction. As the midpoint potential decreased, there was a parallel decrease in the rate of b reduction, demonstrating that the rate of b reduction is dependent upon the rate of ubiquinol oxidation by the iron-sulfur protein. Together these results indicate that ubiquinol oxidation is a concerted reaction in which both the low potential and high potential redox components control ubiquinol oxidation at center P, consistent with the protonmotive Q cycle mechanism.     

Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria

Much evidence indicates that superoxide is generated from O2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O2, little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H2O2-peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H2O2, which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H2O2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O2. Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O2. These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O2 to O2-.     

The antimycin-A-insensitive respiratory pathway of Candida parapsilosis: evidence for a second quinone involved specifically in its functioning

The involvement of a quinone in the antimycin A-insensitive electron transfer from NADH-dehydrogenase to cytochrome c via the alternative respiratory chain of Candida parapsilosis, by-passing complex II, has been studied. After a partial extraction of quinones, the residual respiration was fully antimycin-A-sensitive, but reincorporation of the organic extract partially restored an antimycin A-insensitive respiration. Analysis of quinone content by HPLC, after purification by thin-layer chromatography, evidenced another quinone species in a very low amount. Myxothiazol and stigmatellin were shown to inhibit the alternative pathway but at a higher concentration than required to inhibit the classical pathway. Cytochrome spectra analysis showed that, in the presence of high myxothiazol concentrations, cytochromes c and aa3 were not reduced, while they were in the presence of antimycin A. It is suggested that the secondary pathway of C. parapsilosis involved a specific quinone pool which can be displaced from its binding site by high concentrations of myxothiazol or analogous compounds.     

Multiple Q-cycle bypass reactions at the Qo site of the cytochrome bc1 complex

The cytochrome (cyt) bc(1) complex is central to energy transduction in many species. Most investigators now accept a modified Q-cycle as the catalytic mechanism of this enzyme. Several thermodynamically favorable side reactions must be minimized for efficient functioning of the Q-cycle. Among these, reduction of oxygen by the Q(o) site semiquinone to produce superoxide is of special pathobiological interest. These superoxide-producing bypass reactions are most notably observed as the antimycin A- or myxothiazol-resistant reduction of cyt c. In this work, we demonstrate that these inhibitor-resistant cyt c reductase activities are largely unaffected by removal of O(2) in the isolated yeast cyt bc(1) complex. Further, increasing O(2) tension 5-fold stimulated the antimycin A-resistant reduction by a small amount ( approximately 25%), while leaving the myxothiazol-resistant reduction unchanged. This most likely indicates that the rate-limiting step in superoxide production is the formation of a reactive species (probably a semiquinone), capable of rapid O(2) reduction, and that in the absence of O(2) this species can reduce cyt c by some other pathway. We suggest as one possibility that a semiquinone escapes from the Q(o) site and reduces either O(2) or cyt c directly. The small increase in antimycin A-resistant cyt c reduction rate at high O(2) can be explained by the accumulation of a low concentration of a semiquinone inside the Q(o) site. Under aerobic conditions, addition of saturating levels of superoxide dismutase (SOD) inhibited 50% of cyt c reduction in the presence of myxothiazol, implying that essentially all bypass reactions occur with the production of superoxide. However, SOD inhibited only 35% of antimycin A-resistant cyt c reduction, suggesting the presence of a second, slower bypass reaction that does not reduce O(2). Given that myxothiazol blocks cyt b reduction whereas antimycin A promotes it, we propose that this second bypass occurs by reduction of the Q(o) site semiquinone by prereduced cyt b(L).     

Identification of a stable ubisemiquinone and characterization of the effects of ubiquinone oxidation-reduction status on the Rieske iron-sulfur protein in the three-subunit ubiquinol-cytochrome c oxidoreductase complex of Paracoccus denitrificans

The ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex from Paracoccus denitrificans exhibits a thermodynamically stable ubisemiquinone radical detectable by EPR spectroscopy. The radical is centered at g = 2.004, is sensitive to antimycin, and has a midpoint potential at pH 8.5 of +42 mV. These properties are very similar to those of the stable ubisemiquinone (Qi) previously characterized in the cytochrome bc1 complexes of mitochondria. The micro-environment of the Rieske iron-sulfur cluster in the Paracoccus cytochrome bc1 complex changes in parallel with the redox state of the ubiquinone pool. This change is manifested as shifts in the gx, gy, and gz values of the iron-sulfur cluster EPR signal from 1.80, 1.89, and 2.02 to 1.76, 1.90, and 2.03, respectively, as ubiquinone is reduced to ubiquinol. The spectral shift is accompanied by a broadening of the signal and follows a two electron reduction curve, with a midpoint potential at pH 8.5 of +30 mV. A hydroxy analogue of ubiquinone, UHDBT, which inhibits respiration in the cytochrome bc1 complex, shifts the gx, gy, and gz values of the iron-sulfur cluster EPR signal to 1.78, 1.89, and 2.03, respectively, and raises the midpoint potential of the iron-sulfur cluster at pH 7.5 from +265 to +320 mV. These changes in the micro-environment of the Paracoccus Rieske iron-sulfur cluster are like those elicited in mitochondria. These results indicate that the cytochrome bc1 complex of P. denitrificans has a binding site for ubisemiquinone and that this site confers properties on the bound ubisemiquinone similar to those in mitochondria. In addition, the line shape of the Rieske iron-sulfur cluster changes in response to the oxidation-reduction status of ubiquinone, and the midpoint of the iron-sulfur cluster increases in the presence of a hydroxyquinone analogue of ubiquinone. The latter results are also similar to those observed in the mitochondrial cytochrome bc1 complex. However, unlike the mitochondrial complexes, which contain eight to 11 polypeptides and are thought to contain distinct quinone binding proteins, the Paracoccus cytochrome bc1 complex contains only three polypeptide subunits, cytochromes b, c1, and iron-sulfur protein. The ubisemiquinone binding site and the site at which ubiquinone and/or ubiquinol bind to affect the Rieske iron-sulfur cluster in Paracoccus thus exist in the absence of any distinct quinone binding proteins and must be composed of domains contributed by the cytochromes and/or iron-sulfur protein.     

Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans

A ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex has been purified from the plasma membrane of aerobically grown Paracoccus denitrificans by extraction with dodecyl maltoside and ion exchange chromatography of the extract. The purified complex contains two spectrally and thermodynamically distinct b cytochromes, cytochrome c1, and a Rieske-type iron-sulfur protein. Optical spectra indicate absorption peaks at 553 nm for cytochrome c1 and at 560 and 566 nm for the high and low potential hemes of cytochrome b. The spectrum of cytochrome b560 is shifted to longer wavelength by antimycin. The Paracoccus bc1 complex consists of only three polypeptide subunits. On the basis of their relative electrophoretic mobilities, these have apparent molecular masses of 62, 39, and 20 kDa. The 62- and 39-kDa subunits have been identified as cytochromes c1 and b, respectively. The 20-kDa subunit is assumed to be the Rieske-type iron-sulfur protein on the basis of its molecular weight and the presence of an EPR-detectable signal typical of this iron-sulfur protein in the three-subunit complex. The Paracoccus bc1 complex catalyzes reduction of cytochrome c by ubiquinol with a turnover of 470 s-1. This activity is inhibited by antimycin, myxothiazol, stigmatellin, and hydroxyquinone analogues of ubiquinone, all of which inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain. The electron transfer functions of the Paracoccus complex thus appear to be similar, and possibly identical, to those of the bc1 complex of eukaryotic mitochondria. The Paracoccus bc1 complex has the simplest subunit composition and one of the highest turnover numbers of any bc1 complex isolated from any species to date. These properties suggest that the structural requirements for electron transfer from ubiquinol to cytochrome c are met by a small number of peptides and that the "extra" peptides occurring in the mitochondrial bc1 complexes serve some other function(s), possibly in biogenesis or insertion of the complex into that organelle.     

Effects of dibromothymoquinone on the structure and function of the mitochondrial bc1 complex

We have investigated in detail the effects of dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on the ubiquinol-cytochrome c reductase (cytochrome bc1 complex) from bovine heart mitochondria. The inhibitory action of DBMIB on the steady-state activity of the bc1 complex is related to the specific binding of the quinone to the purified enzymatic complex. At concentrations higher than 10 mol per mol of the enzyme, DBMIB is able to stimulate an antimycin-insensitive reduction of cytochrome c catalyzed by the bc1 complex. In accordance with kinetic data showing a competition by endogenous ubiquinone in the inhibitory action, DBMIB can be considered as a product-like inhibitor of the ubiquinol-cytochrome c reductase activity. The site of specific binding of dibromothymoquinone in the bc1 complex enables it to interact with the iron-sulphur center of the enzyme, as indicated by changes induced in the EPR spectrum of the center. However, the inhibitor also directly interacts with cytochrome b, promoting a fast chemical oxidation of the reduced heme center. In spite of these effects, DBMIB has been found not to exert significant effects on the first turnover of the fully oxidized bc1 complex, as monitored by the rapid reduction of both cytochromes b and c1 by ubiquinol-1. In the presence of antimycin, only a stimulation of cytochrome c1 reduction, in parallel to an enhanced cytochrome b reoxidation, is observed. Moreover, DBMIB does not affect the oxidant-induced extra cytochrome b reduction in the presence of antimycin. On the basis of the evidences suggesting a competition with the endogenous ubiquinone in the redox cycle of the bc1 complex, a model is proposed for the mechanism of DBMIB inhibition. Such model can also explain at the molecular level the redox bypass induced by dibromothymoquinone in the whole respiratory chain (Degli Esposti, M., Rugolo, M. and Lenaz, G. (1983) FEBS Lett. 156, 15-19).     

Dimeric ubiquinol:cytochrome c reductase of Neurospora mitochondria contains one cooperative ubiquinone-reduction centre

Dimeric ubiquinol:cytochrome c reductase of Neurospora mitochondria was isolated as a protein-Triton complex and free of ubiquinol (Q). The enzyme was incorporated into phosphatidylcholine membranes together with Q. The effects of varying the molar ratio of Q to enzyme on the electron transfer from duroquinol (DHQ2) to the cytochromes c, c1 and b were studied. The rate of electron flow from DQH2 to cytochrome c was 15 times increased by Q and was maximal when one molecule of Q was bound to one enzyme dimer. The apparent Km value for DQH2 of the Q-free enzyme was 5 microM and of the Q-supplemented enzyme 25 microM. The pre-steady-state rate of electron transfer from DQH2 to cytochrome c1 was also 15 times increased by Q and was maximal with one Q molecule bound to one enzyme dimer. This effect of Q was inhibited by antimycin. The pre-steady-state rate of electron transfer from DQH2 to cytochrome b was 5 times decreased when Q was bound to the enzyme and this effect of Q was insensitive to myxothiazol. The H+/2e- stoichiometry with DQH2 as substrate of the Q-supplemented enzyme was 3.6. These results are interpreted in accordance with a Q-cycle mechanism operating in a dimeric cytochrome reductase. Each enzyme monomer catalyses a single electron transfer from the QH2-oxidation centre to the Q-reduction centre and the two monomers cooperate in the reduction of Q to QH2 at one Q-reduction centre. This centre contains two different binding sites for Q. DQH2 does not properly react at the QH2-oxidation centre. DQH2, however, binds to the loose Q-binding site of the Q-reduction centre and reduces the Q bound to the tight Q-binding site of the centre. The QH2 thus formed at the Q-reduction centre serves as electron donor for the QH2-oxidation centre.     

EPR characterization of the cytochrome b-c1 complex from Rhodobacter sphaeroides

EPR characteristics of cytochrome c1, cytochromes b-565 and b-562, the iron-sulfur cluster, and an antimycin-sensitive ubisemiquinone radical of purified cytochrome b-c1 complex of Rhodobacter sphaeroides have been studied. The EPR specra of cytochrome c1 shows a signal at g = 3.36 flanked with shoulders. The oxidized form of cytochrome b-562 shows a broad EPR signal at g = 3.49, while oxidized cytochrome b-565 shows a signal at g = 3.76, similar to those of two b cytochromes in the mitochondrial complex. The distribution of cytochromes b-565 and b-562 in the isolated complex is 44 and 56%, respectively. Antimycin and 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB) have little effect on the g = 3.76 signal, but they cause a slight downfield and upfield shifts of the g = 3.49 signal, respectively. 5-Undecyl-6-hydroxyl-4,7-dioxobenzothiazole (UHDBT) shifts the g = 3.49 signal downfield to g = 3.56 and sharpens the g = 3.76 signal slightly. Myxothiazol causes an upfield shift of both g = 3.49 and g = 3.76 signals. EPR characteristics of the reduced iron-sulfur cluster in bacterial cytochrome b-c1 complex are: gx = 1.8 with a small shoulder at g = 1.76, gy = 1.89 and gz = 2.02, similar to those observed with the mitochondrial enzyme. The gx = 1.8 signal decreased and the shoulder increased concurrently as the redox potential decreased, indicating that the environment of the iron-sulfur cluster is sensitive to the redox state of the complex. UHDBT sharpens the gz and and shifts it downfield from g = 2.02 to 2.03, and shifts gx upfield from g = 1.80 to 1.78. UHDBT also causes an upfield shift of gy but to a much lesser extent compared to the other two signals. Addition of DBMIB causes a downfield shift of the gy from 1.89 to 1.94 and broadens the gx signal with an upfield to g = 1.75. Myxothiazol and antimycin show little effect on the gy and gz signals, but they broaden and shift the gx signal upfield to g = 1.74. However, the myxothiazol effect is partially reversed by UHDBT. An antimycin-sensitive ubisemiquinone radical was detected in the cytochrome b-c1 complex. At pH 8.4, the antimycin-sensitive ubisemiquinone radical has a maximal concentration of 0.66 mol per mol complex at 100 mV.(ABSTRACT TRUNCATED AT 400 WORDS)