Elimination of the disulfide bridge in the Rieske iron-sulfur protein allows assembly of the [2Fe-2S] cluster into the Rieske protein but damages the ubiquinol oxidation site in the cytochrome bc1 complex

The [2Fe-2S] cluster of the Rieske iron-sulfur protein is held between two loops of the protein that are connected by a disulfide bridge. We have replaced the two cysteines that form the disulfide bridge in the Rieske protein of Saccharomyces cerevisiae with tyrosine and leucine, and tyrosine and valine, to evaluate the effects of the disulfide bridge on assembly, stability, and thermodynamic properties of the Rieske iron-sulfur cluster. EPR spectra of the Rieske proteins lacking the disulfide bridge indicate the iron-sulfur cluster is assembled in the absence of the disulfide bridge, but there are significant shifts in all g values, indicating a change in the electronic structure of the [2Fe-2S] iron-sulfur center. In addition, the midpoint potential of the iron-sulfur cluster is lowered from 265 mV in the Rieske protein from wild-type yeast to 150 mV in the protein from the C164Y/C180L mutant and to 160 mV in the protein from the C164Y/C180V mutant. Ubiquinol-cytochrome c reductase activities of the bc(1) complexes with Rieske proteins lacking the disulfide bridge are less than 1% of the activity of the bc(1) complex from wild-type yeast, even though normal amounts of the iron-sulfur protein are present as judged by Western blot analysis. These activities are lower than the 105-115 mV decrease in the midpoint potential of the Rieske iron-sulfur cluster can account for. Pre-steady-state reduction of the bc(1) complexes with menadiol indicates that quinol is not oxidized through center P but is oxidized through center N. In addition, the levels of stigmatellin and UHDBT binding are markedly diminished, while antimycin binding is unaffected, in the bc(1) complexes with Rieske proteins lacking the disulfide bridge. Taken together, these results indicate that the ubiquinol oxidation site at center P is damaged in the bc(1) complexes with Rieske proteins lacking the disulfide bridge even though the iron-sulfur cluster is assembled into the Rieske protein.     

Anti-cooperative oxidation of ubiquinol by the yeast cytochrome bc1 complex

We have investigated the interaction between monomers of the dimeric yeast cytochrome bc(1) complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in cytochrome c(1) reduction. At pH 8.8, where the redox potential of the iron-sulfur protein is approximately 200 mV and in a bc(1) complex with a mutated iron-sulfur protein of equally low redox potential, the amount of cytochrome c(1) reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bc(1) complex. Similar experiments in the presence of several equivalents of cytochrome c also showed only half of the bc(1) complex participating in quinol oxidation. The extent of cytochrome b reduced corresponded to two b(H) hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two cytochrome b subunits. Antimycin stimulated the ubiquinol-cytochrome c reductase activity of the bc(1) complex at low inhibitor/enzyme ratios. This stimulation could only be fitted to a model in which half of the bc(1) dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the cytochrome b subunits of the dimer. These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bc(1) complex dimer.     

Regulatory interactions between ubiquinol oxidation and ubiquinone reduction sites in the dimeric cytochrome bc1 complex

We have obtained evidence for conformational communication between ubiquinol oxidation (center P) and ubiquinone reduction (center N) sites of the yeast bc1 complex dimer by analyzing antimycin binding and heme bH reduction at center N in the presence of different center P inhibitors. When stigmatellin was occupying center P, concentration-dependent binding of antimycin occurred only to half of the center N sites. The remaining half of the bc1 complex bound antimycin with a slower rate that was independent of inhibitor concentration, indicating that a slow conformational change needed to occur before half of the enzyme could bind antimycin. In contrast, under conditions where the Rieske protein was not fixed proximal to heme bL at center P, all center N sites bound antimycin with fast and concentration-dependent kinetics. Additionally, the extent of fast cytochrome b reduction by menaquinol through center N in the presence of stigmatellin was approximately half of that observed when myxothiazol was bound at center P. The reduction kinetics of the bH heme by decylubiquinol in the presence of stigmatellin or myxothiazol were also consistent with a model in which fixation of the Rieske protein close to heme bL in both monomers allows rapid binding of ligands only to one center N. Decylubiquinol at high concentrations was able to abolish the biphasic binding of antimycin in the presence of stigmatellin but did not slow down antimycin binding rates. These results are discussed in terms of half-of-the-sites activity of the dimeric bc1 complex.     

The iron-sulfur cluster of the Rieske iron-sulfur protein functions as a proton-exiting gate in the cytochrome bc(1) complex

The destruction of the Rieske iron-sulfur cluster ([2Fe-2S]) in the bc(1) complex by hematoporphyrin-promoted photoinactivation resulted in the complex becoming proton-permeable. To study further the role of this [2Fe-2S] cluster in proton translocation of the bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with mutations at the histidine ligands of the [2Fe-2S] cluster were generated and characterized. These mutants lacked the [2Fe-2S] cluster and possessed no bc(1) activity. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc(1) complex or cytochrome c oxidase, the proton ejection, normally observed in intact reductase or oxidase vesicles during the oxidation of their corresponding substrates, disappeared. This indicated the creation of a proton-leaking channel in the mutant complex, whose [2Fe-2S] cluster was lacking. Insertion of the bc(1) complex lacking the head domain of the Rieske iron-sulfur protein, removed by thermolysin digestion, into PL vesicles together with mitochondrial bc(1) complex also rendered the vesicles proton-permeable. Addition of the excess purified head domain of the Rieske iron-sulfur protein partially restored the proton-pumping activity. These results indicated that elimination of the [2Fe-2S] cluster in mutant bc(1) complexes opened up an otherwise closed proton channel within the bc(1) complex. It was speculated that in the normal catalytic cycle of the bc(1) complex, the [2Fe-2S] cluster may function as a proton-exiting gate.     

Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. III. Import, protease processing, and assembly into the cytochrome bc1 complex of iron-sulfur protein lacking the iron-sulfur cluster.

We have used site-directed mutagenesis of the Saccharomyces cerevisiae Rieske iron-sulfur protein gene (RIP 1) to convert cysteines 159, 164, 178, and 180 to serines, and to convert histidines 161 and 181 to arginines. These 4 cysteines and 2 histidines are conserved in all Rieske proteins sequenced to date, and 4 of these 6 residues are thought to ligate the iron-sulfur cluster to the apoprotein. We have also converted histidine 184 to arginine. This histidine is conserved only in respiring organisms. The site-directed mutations of the six fully conserved putative iron-sulfur cluster ligands result in an inactive iron-sulfur protein, lacking iron-sulfur cluster, and failure of the yeast to grow on nonfermentable carbon sources. In contrast, when histidine 184 is replaced by arginine, the iron-sulfur cluster is assembled properly and the yeast grow on nonfermentable carbon sources. The site-directed mutations of the 6 fully conserved residues do not prevent post-translational import of iron-sulfur protein precursor into mitochondria, nor do the mutations prevent processing of iron-sulfur protein precursor to mature size protein by mitochondrial proteases. Optical spectra of mitochondria from the six mutants indicate that cytochrome b is normal, in contrast to the deranged spectrum of cytochrome b which results when the iron-sulfur protein gene is deleted. In addition, mature size iron-sulfur apoprotein is associated with cytochrome bc1 complex purified from a site-directed mutant in which iron-sulfur cluster is not inserted. These results indicate that mature size iron-sulfur apoprotein, lacking iron-sulfur cluster, is inserted into the cytochrome bc1 complex, where it interacts with and preserves the optical properties of cytochrome b. Insertion of the iron-sulfur cluster is not an obligatory prerequisite to processing of the protein to its final size. Either the processing protease cannot distinguish between iron-sulfur protein with or without the iron-sulfur cluster, or insertion of the iron-sulfur cluster occurs after the protein is processed to its mature size, possibly after it is assembled in the cytochrome bc1 complex.     

A concerted,alternating sites mechanism of ubiquinol oxidation by the dimeric cytochrome bc(1) complex

A refinement of the protonmotive Q cycle mechanism is proposed in which oxidation of ubiquinol is a concerted reaction and occurs by an alternating, half-of-the-sites mechanism. A concerted mechanism of ubiquinol oxidation is inferred from the finding that there is reciprocal control between the high potential and low potential redox components involved in ubiquinol oxidation. The potential of the Rieske iron-sulfur protein controls the rate of reduction of the b cytochromes, and the potential of the b cytochromes controls the rate of reduction of the Rieske protein and cytochrome c(1). A concerted mechanism of ubiquinol oxidation reconciles the findings that the ubiquinol-cytochrome c reductase kinetics of the bc(1) complex include both a pH dependence and a dependence on Rieske iron-sulfur protein midpoint potential.An alternating, half-of-the-sites mechanism for ubiquinol oxidation is inferred from the finding that some inhibitory analogs of ubiquinol that block ubiquinol oxidation by binding to the ubiquinol oxidation site in the bc(1) complex inhibit the yeast enzyme with a stoichiometry of 0.5 per bc(1) complex. One molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme, and the binding is anti-cooperative, in that a second molecule of inhibitor binds with much lower affinity to a dimer in which an inhibitor molecule is already bound. An alternating, half-of-the-sites mechanism implies that, at least under some conditions, only half of the sites in the dimeric enzyme are reactive at any one time. This provides a raison d'être for the dimeric structure of the enzyme, in that bc(1) activity may be regulated and capable of switching between a half-of-the-sites active and a fully active enzyme.     

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.     

Proton translocation by the cytochrome bc1 complexes of phototrophic bacteria: introducing the activated Q-cycle.

The cytochrome bc1 complexes are proton-translocating, dimeric membrane ubiquinol:cytochrome c oxidoreductases that serve as "hubs" in the vast majority of electron transfer chains. After each ubiquinol molecule is oxidized in the catalytic center P at the positively charged membrane side, the two liberated electrons head out, according to the Mitchell's Q-cycle mechanism, to different acceptors. One is taken by the [2Fe-2S] iron-sulfur Rieske protein to be passed further to cytochrome c1. The other electron goes across the membrane, via the low- and high-potential hemes of cytochrome b, to another ubiquinone-binding site N at the opposite membrane side. It has been assumed that two ubiquinol molecules have to be oxidized by center P to yield first a semiquinone in center N and then to reduce this semiquinone to ubiquinol. This review is focused on the operation of cytochrome bc1 complexes in phototrophic purple bacteria. Their membranes provide a unique system where the generation of membrane voltage by light-driven, energy-converting enzymes can be traced via spectral shifts of native carotenoids and correlated with the electron and proton transfer reactions. An "activated Q-cycle" is proposed as a novel mechanism that is consistent with the available experimental data on the electron/proton coupling. Under physiological conditions, the dimeric cytochrome bc1 complex is suggested to be continually primed by prompt oxidation of membrane ubiquinol via center N yielding a bound semiquinone in this center and a reduced, high-potential heme b in the other monomer of the enzyme. Then the oxidation of each ubiquinol molecule in center P is followed by ubiquinol formation in center N, proton translocation and generation of membrane voltage.     

Intermediate length Rieske iron-sulfur protein is present and functionally active in the cytochrome bc1 complex of Saccharomyces cerevisiae

To investigate the relationship between post-translational processing of the Rieske iron-sulfur protein of Saccharomyces cerevisiae and its assembly into the mitochondrial cytochrome bc1 complex we used iron-sulfur proteins in which the presequences had been changed by site-directed mutagenesis of the cloned iron-sulfur protein gene, so that the recognition sites for the matrix processing peptidase or the mitochondrial intermediate peptidase (MIP) had been destroyed. When yeast strain JPJ1, in which the gene for the iron-sulfur protein is deleted, was transformed with these constructs on a single copy expression vector, mitochondrial membranes and bc1 complexes isolated from these strains accumulated intermediate length iron-sulfur proteins in vivo. The cytochrome bc1 complex activities of these membranes and bc1 complexes indicate that intermediate iron-sulfur protein (i-ISP) has full activity when compared with that of mature sized iron-sulfur protein (m-ISP). Therefore the iron-sulfur cluster must have been inserted before processing of i-ISP to m-ISP by MIP. When iron-sulfur protein is imported into mitochondria in vitro, i-ISP interacts with components of the bc1 complex before it is processed to m-ISP. These results establish that the iron-sulfur cluster is inserted into the apoprotein before MIP cleaves off the second part of the presequence and that this second processing step takes place after i-ISP has been assembled into the bc1 complex.     

The structure of the soluble domain of an archaeal Rieske iron-sulfur protein at 1.1 A resolution

The first crystal structure of an archaeal Rieske iron-sulfur protein, the soluble domain of Rieske iron-sulfur protein II (soxF) from the hyperthermo-acidophile Sulfolobus acidocaldarius, has been solved by multiple wavelength anomalous dispersion (MAD) and has been refined to 1.1 A resolution. SoxF is a subunit of the terminal oxidase supercomplex SoxM in the plasma membrane of S. acidocaldarius that combines features of a cytochrome bc(1) complex and a cytochrome c oxidase. The [2Fe-2S] cluster of soxF is most likely the primary electron acceptor during the oxidation of caldariella quinone by the cytochrome a(587)/Rieske subcomplex. The geometry of the [2Fe-2S] cluster and the structure of the cluster-binding site are almost identical in soxF and the Rieske proteins from eucaryal cytochrome bc(1) and b(6)f complexes, suggesting a strict conservation of the catalytic mechanism. The main domain of soxF and part of the cluster-binding domain, though structurally related, show a significantly divergent structure with respect to topology, non-covalent interactions and surface charges. The divergent structure of soxF reflects a different topology of the soxM complex compared to eucaryal bc complexes and the adaptation of the protein to the extreme ambient conditions on the outer membrane surface of a hyperthermo-acidophilic organism.     

Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced Rhodobacter capsulatus cytochrome c2 to the cytochrome bc1 complex mediated by the conformation of the Rieske iron-sulfur protein

The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured by plasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor stigmatellin. The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 microM) but binds much more weakly to the oxidized form (Kd = 3.1 microM). In contrast, oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 microM. Such a biphasic interaction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2 and/or cytochrome bc1. However, in the presence of stigmatellin, we find that oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 microM) and reduced cytochrome c2 binds less strongly (Kd = 0.11 microM) but approximately 30-fold more tightly than in the absence of stigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, have led to the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1 components during turnover. In one conformation, the Rieske protein binds near the heme of cytochrome c1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite side from the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor, stigmatellin, freezes the Rieske protein iron-sulfur cluster in a conformation proximal to cytochrome b and distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizes turnover by maximizing binding of the substrate, oxidized cytochrome c2, when the iron-sulfur cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c2, when it is proximal to cytochrome c1.     

The iron-sulfur protein is necessary for the complete assembly of the low-molecular-weight subunits into the cytochrome b-c1 complex of yeast mitochondria

A strain of yeast lacking the gene for the Rieske iron-sulfur protein (RIP) of the cytochrome b-c1 complex was used to study the assembly of this complex in the mitochondrial membrane. This strain lacks the mRNA for the iron-sulfur protein as evidenced by both Northern hybridization using a probe containing the coding region of the gene plus in vitro translation of total RNA followed by immunoprecipitation with a specific antibody against the iron-sulfur protein. In addition, isolated mitochondria from this strain lacked cytochrome c reductase activity with either succinate or the decyl analog of ubiquinol as substrate. Immunoblotting studies with antiserum against the cytochrome b-c1 complex revealed that mitochondria from the iron-sulfur protein-deficient strain have levels of core protein I, core protein II, and cytochrome c1 equal to those of wild-type mitochondria; however, a decrease in cytochrome b was evident from both immunoblotting and spectral analysis. Moreover, it is evident from the immunoprecipitates of radiolabeled mitochondria that the amounts of the low-molecular-weight subunits (17, 14, and 11 kDa) are decreased 53, 65, and 50%, respectively, in mitochondria lacking the iron-sulfur protein. These results suggest that the iron-sulfur protein is required for the complete assembly of the low-molecular-weight subunits into the cytochrome b-c1 complex.     

The LYR protein Mzm1 functions in the insertion of the Rieske Fe/S protein in yeast mitochondria

The assembly of the cytochrome bc(1) complex in Saccharomyces cerevisiae is shown to be conditionally dependent on a novel factor, Mzm1. Cells lacking Mzm1 exhibit a modest bc(1) defect at 30°C, but the defect is exacerbated at elevated temperatures. Formation of bc(1) is stalled in mzm1Δ cells at a late assembly intermediate lacking the Rieske iron-sulfur protein Rip1. Rip1 levels are markedly attenuated in mzm1Δ cells at elevated temperatures. Respiratory growth can be restored in the mutant cells by the overexpression of the Rip1 subunit. Elevated levels of Mzm1 enhance the stabilization of Rip1 through physical interaction, suggesting that Mzm1 may be an important Rip1 chaperone especially under heat stress. Mzm1 may function primarily to stabilize Rip1 prior to inner membrane (IM) insertion or alternatively to aid in the presentation of Rip1 to the inner membrane translocation complex for extrusion of the folded domain containing the iron-sulfur center.     

Orientation of the g-tensor axes of the Rieske subunit in the cytochrome bc1 complex

The orientation of the g-tensors of the Rieske iron-sulfur protein subunit was determined in a single crystal of the bovine mitochondrial cytochrome bc1 complex with stigmatellin in the Qo quinol binding site. The g-tensor principal axes are skewed with respect to the Fe-Fe and S-S atom direction in the 2Fe2S cluster, which is allowed by the lack of rigorous symmetry of the cluster. The asymmetric unit in the crystal is the active dimer, and the g-tensor axes have slightly different orientations relative to the iron-sulfur cluster in the two halves of the dimer. The g approximately 1.79 axis makes an average angle of 30 degrees with respect to the Fe-Fe direction and the g approximately 2.024 axis an average angle of 26 degrees with respect to the S-S direction. This assignment of the g-tensor axis directions indicates that conformations of the Rieske protein are likely the same in the cytochrome bc1 and b6f complexes and that the extent of motion of the Rieske head domain during the catalytic cycle has been highly conserved during evolution of these distantly related complexes.     

Inhibitory analogs of ubiquinol act anti-cooperatively on the Yeast cytochrome bc1 complex. Evidence for an alternating, half-of-the-sites mechanism of ubiquinol oxidation.

The cytochrome bc(1) complex is a dimeric enzyme that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is re-reduced at a second center, referred to as center N. To understand better the mechanism of ubiquinol oxidation, we have examined the interaction of several inhibitory analogs of ubiquinol with the yeast cytochrome bc(1) complex. Stigmatellin and methoxyacrylate stilbene, two inhibitors that block ubiquinol oxidation at center P, inhibit the yeast enzyme with a stoichiometry of 0.5 per bc(1) complex, indicating that one molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme. This stoichiometry was obtained when the inhibitors were titrated in cytochrome c reductase assays and in reactions of quinol with enzyme in which the inhibitors block pre-steady state reduction of cytochrome b. As an independent measure of inhibitor binding, we titrated the red shift in the optical spectrum of ferrocytochrome b with methoxyacrylate stilbene and thus confirmed the results of the inhibition of activity titrations. The titration curves also indicate that the binding is anti-cooperative, in that a second molecule of inhibitor binds with much lower affinity to a dimer in which an inhibitor molecule is already bound. Because these inhibitors bind to the ubiquinol oxidation site in the bc(1) complex, we propose that the yeast cytochrome bc(1) complex oxidizes ubiquinol by an alternating, half-of-the-sites mechanism.     

Assembly of the iron-sulfur protein into the cytochrome b-c1, complex of yeast mitochondria.

The assembly of the iron-sulfur protein into the cytochrome bc1 complex after import and processing of the precursor form into mitochondria in vitro was investigated by immunoprecipitation of the radiolabeled iron-sulfur protein from detergent-solubilized mitochondria with specific antisera. After import in vitro, the labeled mature form of the iron-sulfur protein was immunoprecipitated by antisera against both the iron-sulfur protein and the entire bc1 complex from mitochondria solubilized with either Triton X-100 or dodecyl maltoside. After sodium dodecyl sulfate solubilization of mitochondria, however, the antiserum against the iron-sulfur protein, but not that against the bc1 complex, immunoprecipitated the radiolabeled iron-sulfur protein. These results suggest that in mitochondria the mature form of the iron-sulfur protein is assembled with other subunits of the bc1 complex that are recognized by the antiserum against the bc1 complex. By contrast, the intermediate and precursor forms of the iron-sulfur protein that accumulated in the matrix when proteolytic processing was blocked with EDTA and o-phenanthroline were not efficiently assembled into the bc1 complex. The import and processing of the iron-sulfur protein also occurred in mitochondria lacking either cytochrome b (W-267) or the iron-sulfur protein (JPJ1). The mature form of the iron-sulfur protein was immunoprecipitated by antisera against the bc1 complex or core protein I after import in vitro into these mitochondria, suggesting that the mature form is associated with other subunits of the bc1 complex in these strains.     

The role of the Rieske iron-sulfur center as the electron donor to ferricytochrome c2 in Rhodopseudomonas sphaeroides

The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c2, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation (t 1/2 3-5 microseconds). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (102 ms) antimycin-sensitive phase of ferricytochrome c2 reduction, attributed to a specific ubiquinone-10 molecule (Qz), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Qz (QzH2) reduces the Rieske Center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c2 reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center. UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from gy 1.90 to gy 1.89, and shifts the Em,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c2, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of HII(+); these may reflect additional sites of action of the inhibitor.     

Stigmatellin induces reduction of iron-sulfur protein in the oxidized cytochrome bc1 complex

Stigmatellin, a Q(P) site inhibitor, inhibits electron transfer from iron-sulfur protein (ISP) to cytochrome c1 in the bc1 complex. Stigmatellin raises the midpoint potential of ISP from 290 mV to 540 mV. The binding of stigmatellin to the fully oxidized complex, oxidized completely by catalytic amounts of cytochrome c oxidase and cytochrome c, results in ISP reduction. The extent of ISP reduction is proportional to the amount of inhibitor used and reaches a maximum when the ratio of inhibitor to enzyme complex reaches unity. A g = 2.005 EPR peak, characteristic of an organic free radical, is also observed when stigmatellin is added to the oxidized complex, and its signal intensity depends on the amount of stigmatellin. Addition of ferricyanide, a strong oxidant, to the oxidized complex also generates a g = 2.005 EPR peak that is oxidant concentration-dependent. Oxygen radicals are generated when stigmatellin is added to the oxidized complex in the absence of the exogenous substrate, ubiquinol. The amount of oxygen radical formed is proportional to the amount of stigmatellin added. Oxygen radicals are not generated when stigmatellin is added to a mutant bc1 complex lacking the Rieske iron-sulfur cluster. Based on these results, it is proposed that ISP becomes a strong oxidant upon stigmatellin binding, extracting electrons from an organic compound, likely an amino acid residue. This results in the reduction of ISP and generation of organic radicals.     

Rapid electron transfer between monomers when the cytochrome bc1 complex dimer is reduced through center N

We have obtained evidence for electron transfer between cytochrome b subunits of the yeast bc(1) 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 bc(1) complex dimer. The b(H) heme absorbance in a bc(1) 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 b(H) 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 b(H) hemes in the dimer through the two b(L) hemes is possible upon reduction through one center N despite the thermodynamically unfavorable b(H) to b(L) electron transfer step. We propose that electron transfer between cytochrome b subunits minimizes the formation of semiquinone-ferrocytochrome b(H) complexes at center N and favors ubiquinol oxidation at center P by increasing the amount of oxidized cytochrome b.     

Interaction of stigmatellin and DNP-INT with the Rieske iron-sulfur center of the chloroplast cytochrome b6-f complex

Stigmatellin and DNP-INT are effective inhibitors of the catalytic activity of the plastoquinol-plastocyanin oxidoreductase complex (cytochrome b6-f complex). Both inhibitors alter the EPR spectrum of the Rieske iron-sulfur center but do not produce band-shifts of cytochrome b-563. The midpoint redox potential of the Rieske center is unaffected by either inhibitor, although both alter the DBMIB-induced g-value shifts of the Rieske center. The results are considered in terms of binding domains for inhibitors in the cytochrome b6-f complex.