Reduction potentials of Rieske clusters: importance of the coupling between oxidation state and histidine protonation state

Rieske [2Fe-2S] clusters can be classified into two groups, depending on their reduction potentials. Typical high-potential Rieske proteins have pH-dependent reduction potentials between +350 and +150 mV at pH 7, and low-potential Rieske proteins have pH-independent potentials of around -150 mV at pH 7. The pH dependence of the former group is attributed to coupled deprotonation of the two histidine ligands. Protein-film voltammetry has been used to compare three Rieske proteins: the high-potential Rieske proteins from Rhodobacter sphaeroides (RsRp) and Thermus thermophilus (TtRp) and the low-potential Rieske ferredoxin from Burkholderia sp. strain LB400 (BphF). RsRp and TtRp differ because there is a cluster to serine hydrogen bond in RsRp, which raises its potential by 140 mV. BphF lacks five hydrogen bonds to the cluster and an adjacent disulfide bond. Voltammetry measurements between pH 3 and 14 reveal that all the proteins, including BphF, have pH-dependent reduction potentials with remarkably similar overall profiles. Relative to RsRp and TtRp, the potential versus pH curve of BphF is shifted to lower potential and higher pH, and the pK(a) values of the histidine ligands of the oxidized and reduced cluster are closer together. Therefore, in addition to simple electrostatic effects on E and pK(a), the reduction potentials of Rieske clusters are determined by the degree of coupling between cluster oxidation state and histidine protonation state. Implications for the mechanism of quinol oxidation at the Q(O) site of the cytochrome bc(1) and b(6)f complexes are discussed.     

Resonance Raman characterization of archaeal and bacterial Rieske protein variants with modified hydrogen bond network around the [2Fe-2S] center

The rate of quinol oxidation by cytochrome bc(1)/b(6)f complex is in part associated with the redox potential (E(m)) of its Rieske [2Fe-2S] center, for which an approximate correlation with the number of hydrogen bonds to the cluster has been proposed. Here we report comparative resonance Raman (RR) characterization of bacterial and archaeal high-potential Rieske proteins and their site-directed variants with a modified hydrogen bond network around the cluster. Major differences among their RR spectra appear to be associated in part with the presence or absence of Tyr-156 (in the Rhodobacter sphaeroides numbering) near one of the Cys ligands to the cluster. Elimination of the hydrogen bond between the terminal cysteinyl sulfur ligand (S(t)) and Tyr-Oeta (as with the Y156W variant, which has a modified histidine N(epsilon) pK(a,ox)) induces a small structural bias of the geometry of the cluster and the surrounding protein in the normal coordinate system, and significantly affects some Fe-S(b/t) stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (S(b)) and Ser-Ogamma, which is weak and/or unfavorably oriented for extensive coupling with the Fe-S(b/t) stretching vibrations.     

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.     

Negatively charged residues and hydrogen bonds tune the ligand histidine pKa values of Rieske iron-sulfur proteins

Rieske proteins carry a redox-active iron-sulfur cluster, which is bound by two histidine and two cysteine side chains. The reduction potential of Rieske proteins depends on pH. This pH dependence can be described by two pK(a) values, which have been assigned to the two iron-coordinating histidines. Rieske proteins are commonly grouped into two major classes: Rieske proteins from quinol-oxidizing cytochrome bc complexes, in which the ligand histidines titrate in the physiological pH range, and bacterial ferredoxin Rieske proteins, in which the ligand histidines are protonated at physiological pH. In the study presented here, we have calculated pK(a) values of the cluster ligand histidines using a combined density functional theory/continuum electrostatics approach. Experimental pK(a) values for a bc-type and a ferredoxin Rieske protein could be reproduced. We could identify functionally important differences between the two proteins: hydrogen bonds toward the cluster, which are present in bc-type Rieske proteins, and negatively charged residues, which are present in ferredoxin Rieske proteins. We removed these differences by mutating the proteins in our calculations. The Rieske centers in the mutated proteins have very similar pK(a) values. We thus conclude that the studied structural differences are the main reason for the different pH-titration behavior of the proteins. Interestingly, the shift caused by neutralizing the negative charges in ferredoxin Rieske proteins is larger than the shift caused by removing the hydrogen bonds toward the cluster in bc-type Rieske proteins.     

Breaking and re-forming the disulfide bond at the high-potential,respiratory-type Rieske [2Fe-2S] center of thermus thermophilus: characterization of the sulfhydryl state by protein-film voltammetry

A disulfide bond, adjacent to the [2Fe-2S] cluster, is conserved in all high-potential Rieske proteins from the respiratory and photosynthetic cytochrome bc(1) and b(6)f complexes but is absent from the low-potential, bacterial dioxygenase Rieske proteins. The role of the disulfide is unclear, since cysteine mutants have resulted in only apoprotein. The high stability of the soluble Thermus thermophilus Rieske protein permits chemical reduction of the disulfide bond and characterization of the sulfhydryl (dithiol) form by protein-film voltammetry. The effect of disulfide reduction on the cluster potential is small (DeltaE(0)' 相似文献   

Failure to insert the iron-sulfur cluster into the Rieske iron-sulfur protein impairs both center N and center P of the cytochrome bc1 complex

Mutation of a serine that forms a hydrogen bond to the iron-sulfur cluster of the Rieske iron-sulfur protein to a cysteine results in a respiratory-deficient yeast strain due to formation of iron-sulfur protein lacking the iron-sulfur cluster. The Rieske apoprotein lacking the iron-sulfur cluster is inserted into both monomers of the dimeric cytochrome bc(1) complex and processed to mature size, but the protein lacking iron-sulfur cluster is more susceptible to proteolysis. In addition, the protein environment of center P in one half of the dimer is affected by failure to insert the iron-sulfur cluster as indicated by the fact that only one molecule of myxothiazol can be bound to the cytochrome bc(1) dimer. Although the bc(1) complex lacking the Rieske iron-sulfur cluster cannot oxidize ubiquinol through center P, rates of reduction of cytochrome b by menaquinol through center N are normal. However, less cytochrome b is reduced through center N, and only one molecule of antimycin can be bound at center N in the bc(1) dimer lacking iron-sulfur cluster. These results indicate that failure to insert the [2Fe-2S] cluster impairs assembly of the Rieske protein into the bc(1) complex and that this interferes with proper assembly of both center P and center N in one half of the dimeric enzyme.     

Tight binding of inhibitors to bovine bc1 complex is independent of the Rieske protein redox state. Consequences for semiquinone stabilization in the quinol oxidation site

To determine the effect of the redox state of the Rieske protein on ligand binding to the quinol oxidation site of the bc(1) complex, we measured the binding rate constants (k(1)) for stigmatellin and myxothiazol, at different concentrations of decylbenzoquinone or decylbenzoquinol, in the bovine bc(1) complex with the Rieske protein in the oxidized or reduced state. Stigmatellin and myxothiazol bound tightly and competitively with respect to quinone or quinol, independently of the redox state of the Rieske protein. In the oxidized bc(1) complex, the k(1) values for stigmatellin ( approximately 2.6 x 10(6) m(-1)s(-1)) and myxothiazol ( approximately 8 x 10(5) m(-1)s(-1)), and the dissociation constant (K(d)) for quinone, were similar between pH 6.5 and 9, indicating that ligand binding is independent of the protonation state of histidine 161 of the Rieske protein (pK(a) approximately 7.6). Reduction of the Rieske protein increased the k(1) value for stigmatellin and decreased the K(d) value for quinone by 50%, without modifying the k(1) for myxothiazol. These results indicate that reduction of the Rieske protein and protonation of histidine 161 do not induce a strong stabilization of ligand binding to the quinol oxidation site, as assumed in models that propose the existence of a highly stabilized semiquinone as a reaction intermediate during quinol oxidation.     

Controlling the functionality of cytochrome c(1) redox potentials in the Rhodobacter capsulatus bc(1) complex through disulfide anchoring of a loop and a beta-branched amino acid near the heme-ligating methionine.

The cytochrome c(1) subunit of the ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) contains a single heme group covalently attached to the polypeptide via thioether bonds of two conserved cysteine residues. In the photosynthetic bacterium Rhodobacter (Rba.) capsulatus, cytochrome c(1) contains two additional cysteines, C144 and C167. Site-directed mutagenesis reveals a disulfide bond (rare in monoheme c-type cytochromes) anchoring C144 to C167, which is in the middle of an 18 amino acid loop that is present in some bacterial cytochromes c(1) but absent in higher organisms. Both single and double Cys to Ala substitutions drastically lower the +320 mV redox potential of the native form to below 0 mV, yielding nonfunctional cytochrome bc(1). In sharp contrast to the native protein, mutant cytochrome c(1) binds carbon monoxide (CO) in the reduced form, indicating an opening of the heme environment that is correlated with the drop in potential. In revertants, loss of the disulfide bond is remediated uniquely by insertion of a beta-branched amino acid two residues away from the heme-ligating methionine 183, identifying the pattern betaXM, naturally common in many other high-potential cytochromes c. Despite the unrepaired disulfide bond, the betaXM revertants are no longer vulnerable to CO binding and restore function by raising the redox potential to +227 mV, which is remarkably close to the value of the betaXM containing but loop-free mitochondrial cytochrome c(1). The disulfide anchored loop and betaXM motifs appear to be two independent but nonadditive strategies to control the integrity of the heme-binding pocket and raise cytochrome c midpoint potentials.     

Abundance, subunit composition, redox properties, and catalytic activity of the cytochrome bc1 complex from alkaliphilic and halophilic, photosynthetic members of the family Ectothiorhodospiraceae.

Ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complexes were demonstrated to be present in the membranes of the alkaliphilic and halophilic purple sulfur bacteria Ectothiorhodospira halophila, Ectothiorhodospira mobilis, and Ectothiorhodospira shaposhnikovii by protoheme extraction, immunoblotting, and electron paramagnetic resonance spectroscopy. The gy values of the Rieske [2Fe-2S] clusters observed in membranes of E. mobilis and E. halophila were 1.895 and 1.910, respectively. In E. mobilis membranes, the cytochrome bc1 complex was present in a stoichiometry of approximately 0.2 per reaction center. This complex was isolated and characterized. It contained four prosthetic groups: low-potential cytochrome b (cytochrome bL; Em = -142 mV), high-potential cytochrome b (cytochrome bH; Em = 116 mV), cytochrome c1 (Em = 341 mV), and a Rieske iron-sulfur cluster. The absorbance spectrum of cytochrome bL displayed an asymmetric alpha-band with a maximum at 564 nm and a shoulder at 559 nm. The alpha bands of cytochrome bH and cytochrome c1 peaked at 559.5 and 553 nm, respectively. These prosthetic groups were associated with three different polypeptides: cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein, with apparent molecular masses of 43, 30, and 21 kDa, respectively. No evidence for the presence of a fourth subunit was obtained. Maximal ubiquinol-cytochrome c oxidoreductase activity of the purified complex was observed at pH 8; the turnover rate was 57 mol of cytochrome c reduced.(mol of cytochrome c1)-1.s-1. The complex showed a strikingly low sensitivity towards typical inhibitors of cytochrome bc1 complexes.     

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.     

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.     

Steered molecular dynamics simulation of the Rieske subunit motion in the cytochrome bc(1) complex.

Crystallographic structures of the mitochondrial ubiquinol/cytochrome c oxidoreductase (cytochrome bc(1) complex) suggest that the mechanism of quinol oxidation by the bc(1) complex involves a substantial movement of the soluble head of the Rieske iron-sulfur protein (ISP) between reaction domains in cytochrome b and cytochrome c(1) subunits. In this paper we report the results of steered molecular dynamics simulations inducing, through an applied torque within 1 ns, a 56 degrees rotation of the soluble domain of ISP. For this purpose, a solvated structure of the bc(1) complex in a phospholipid bilayer (a total of 206,720 atoms) was constructed. A subset of 91,061 atoms was actually simulated with 45,131 moving atoms. Point charge distributions for the force field parametrization of heme groups and the Fe(2)S(2) cluster of the Rieske protein included in the simulated complex were determined. The simulations showed that rotation of the soluble domain of ISP is actually feasible. Several metastable conformations of the ISP during its rotation were identified and the interactions stabilizing the initial, final, and intermediate positions of the soluble head of the ISP domain were characterized. A pathway for proton conduction from the Q(o) site to the solvent via a water channel has been identified.     

Confirmation of the involvement of protein domain movement during the catalytic cycle of the cytochrome bc1 complex by the formation of an intersubunit disulfide bond between cytochrome b and the iron-sulfur protein

To study the essentiality of head domain movement of the Rieske iron-sulfur protein (ISP) during bc(1) catalysis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with three pairs of cysteines engineered (one cysteine each) on the interface between cytochrome b and ISP, A185C(cytb)/K70C(ISP), I326C(cytb)/G165C(ISP), and T386C(cytb)/K164C(ISP), were generated and characterized. Formation of an intersubunit disulfide bond between cytochrome b and ISP is detected in membrane (intracytoplasmic membrane and air-aged chromatophore), and purified bc(1) complex was prepared from the A185C(cytb)/K70C(ISP) mutant cells. Formation of the intersubunit disulfide bond in this cysteine pair mutant complex is concurrent with the loss of its bc(1) activity. Reduction of this disulfide bond by beta-mercaptoethanol restores activity, indicating that mobility of the head domain of ISP is functionally important in the cytochrome bc(1) complex. The rate of intramolecular electron transfer, between 2Fe2S and heme c(1), in the A185C(cytb)/K70C(ISP) mutant complex is much lower than that in the wild type or in their respective single cysteine mutant complexes, indicating that formation of an intersubunit disulfide bond between cytochrome b and ISP arrests the head domain of ISP in the "fixed state" position, which is too far for electron transfer to heme c(1).     

Specific mutagenesis of the rieske iron-sulfur protein in Rhodobacter sphaeroides shows that both the thermodynamic gradient and the pK of the oxidized form determine the rate of quinol oxidation by the bc(1) complex

In the Rieske iron-sulfur protein (ISP) of the ubiquinol:cytochrome c(2) oxidoreductase (bc(1) complex) of Rhodobacter sphaeroides, residue Tyr 156 is located close to the iron-sulfur cluster. Previous studies of the equivalent residue in both Saccharomyces cerevisiae [Denke, E., Merbitz-Zahradnik, T., Hatzfeld, O. M., Snyder, C. H., Link, T. A., and Trumpower, B. L. (1998) J. Biol. Chem. 273, 9085-9093] and Paracoccus denitrificans [Schroter, T., Hatzfeld, O. M., Gemeinhardt, S., Korn, M., Friedrich, T., Ludwig, B. , and Link, T. A. (1998) Eur. J. Biochem. 255, 100-106] have indicated that mutations at this site can lead to modifications in the redox potential of the ISP. To study the effect of similar modifications on the thermodynamic behavior and kinetics of partial reactions of the bc(1) complex upon flash activation, we have constructed four mutant strains of Rb. sphaeroides where Tyr 156 was mutated to His, Leu, Phe, or Trp. The bc(1) complex was assembled and able to support photosynthetic growth in all mutants. Three substitutions (Leu, Phe, Trp) led to alteration of the midpoint potential (E(m)) of the ISP and a slowing in rate of quinol oxidation, suggesting that electron transfer from quinol to the oxidized ISP controls the overall rate and that this step includes the high activation barrier. The Trp mutation led to an increase of approximately 1 pH unit in the pK value of the oxidized ISP. The pH dependence of the rate of quinol oxidation in this mutant was also shifted up by approximately 1 pH unit, showing the importance of the protonation state of the ISP for quinol oxidation. This provides support for a model in which the dissociated form of the oxidized ISP is required for formation of the enzyme-substrate complex [Ugulava, N., and Crofts, A. R. (1998) FEBS Lett. 440, 409-413].     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc1 complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c1 position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c1 [S141C(ISP)/G180C(cyt c1)]. The formation of a disulfide bond between ISP and cyt c1 in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with beta-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc1 complex. Formation of the disulfide bond between ISP and cyt c1 shortens the distance between the [2Fe-2S] cluster and heme c1, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.     

Redox components of cytochrome bc-type enzymes in acidophilic prokaryotes. II. The Rieske protein of phylogenetically distant acidophilic organisms.

The Rieske proteins of two phylogenetically distant acidophilic organisms, i.e. the proteobacterium Thiobacillus ferrooxidans and the crenarchaeon Sulfolobus acidocaldarius, were studied by EPR. Redox titrations at a range of pH values showed that the Rieske centers of both organisms are characterized by redox midpoint potential-versus-pH curves featuring a common pK value of 6.2. This pK value is significantly more acidic (by almost 2 pH units) than that of Rieske proteins in neutrophilic species. The orientations of the Rieske center's g tensors with respect to the plane of the membrane were studied between pH 4 and 8 using partially ordered samples. At pH 4, the Sulfolobus Rieske cluster was found in the "typical" orientation of chemically reduced Rieske centers, whereas this orientation changed significantly on going toward high pH values. The Thiobacillus protein, by contrast, appeared to be in the "standard" orientation at both low and high pH values. The results are discussed with respect to the molecular parameters conveying acid resistance and in light of the recently demonstrated long-range conformational movement of the Rieske protein during enzyme turnover in cytochrome bc1 complexes.     

The Rhodospirillum rubrum cytochrome bc1 complex: peptide composition, prosthetic group content and quinone binding

A cytochrome bc1 complex, essentially free of bacteriochlorophyll, has been purified from the photosynthetic purple non-sulfur bacterium Rhodospirillum rubrum. The complex catalyzes electron flow from quinol to cytochrome c (turnover number = 75 s-1) that is inhibited by low concentrations of antimycin A and myxothiazol. The complex contains only three peptide subunits: cytochrome b (Mr = 35,000); cytochrome c1 (Mr = 31,000) and the Rieske iron-sulfur protein (Mr = 22,400). Em values (pH 7.4) were measured for cytochrome c1 (+320 mV) and the two hemes of cytochrome b (-33 and -90 mV). Electron flow from quinol to cytochrome c is inhibited when the complex is pre-illuminated in the presence of a ubiquinone photoaffinity analog (azido-Q). During illumination, the azido-Q becomes covalently attached to the cytochrome b peptide and, to a lesser extent, to cytochrome c1.     

Protonmotive Q cycle pathway of electron transfer and energy transduction in the three-subunit ubiquinol-cytochrome c oxidoreductase complex of Paracoccus denitrificans

Ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex from Paracoccus denitrificans consists of only three polypeptide subunits (Yang, X., and Trumpower, B. L. (1986) J. Biol. Chem. 261, 12282-12289), whereas the analogous complexes of eukaryotic mitochondria consist of nine or more polypeptides (Schagger, H., Link, T. A., Engel, W. D., and von Jagow, G. (1986) Methods Enzymol. 126, 224-237). Using the purified three-subunit Paracoccus complex we have tested whether this simple cytochrome bc1 complex has the same electron transfer pathway and proton translocation activity as the bc1 complexes of mitochondria. Under presteady state conditions, the effects of inhibitors on reduction of cytochromes b and c1 by quinol and oxidant-induced reduction of cytochrome b indicate a cyclic electron transfer pathway and two routes of cytochrome b reduction in the three-subunit Paracoccus cytochrome bc1 complex. A novel method was developed to incorporate the cytochrome bc1 complex into liposomes with the detergent dodecyl maltoside. The enzyme reconstituted into liposomes translocated protons with an H+/2e value of 3.9. Carbonyl cyanide m-chlorophenylhydrazone eliminated proton translocation, while permitting the scalar release of protons from quinol, and thus reduced the H+/2e ratio to 2. These values agree with the predicted stoichiometries for proton translocation by a protonmotive Q cycle pathway. No inhibition of proton translocation by N',N'-dicyclohexylcarbodiimide was detected when the Paracoccus cytochrome bc1 complex was incubated with N',N'-dicyclohexylcarbodiimide before or after reconstitution into liposomes. Electron transfer in the three-subunit complex thus appears to occur by a protonmotive Q cycle pathway identical to that in mitochondrial cytochrome bc1 complexes. Only three polypeptides, cytochromes b, c1, and the Rieske iron-sulfur protein, are required for respiration and energy transduction in the cytochrome bc1 complex. The function of the supernumerary polypeptides in mitochondrial bc1 complexes is thus unclear.     

On the oxidation pathways of the mitochondrial bc1 complex from beef heart. Effects of various inhibitors

We have investigated the oxidation of the reduced ubiquinol:cytochrome c reductase (bc1 complex) isolated from beef heart mitochondria. The oxidation of cytochrome c1 by both potassium ferricyanide and cytochrome c in the ascorbate-reduced bc1 complex is not a first-order process. This is taken as evidence that cytochrome c1 is in rapid equilibrium with the Rieske iron-sulphur center. Among the several inhibitors tested, only 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole and stigmatellin are seen to affect this redox equilibrium between the high-potential centers of the beef heart bc1 complex. The oxidation of cytochrome b by cytochrome c in both the succinate-reduced and the fully reduced bc1 complex is blocked by all the inhibitors tested. This inhibition occurs simultaneously with an acceleration in the oxidation of cytochrome c1, even after extraction of the endogenous ubiquinone which is present in the bc1 preparation. Almost complete extraction of ubiquinone from the bc1 complex has no effect upon the rapid phase of cytochrome b oxidation, nor does it alter the inhibition of cytochrome b oxidation by the various inhibitors. The oxidation of cytochrome b by exogenous ubiquinones is stimulated by myxothiazol and partially inhibited by antimycin. However, the addition of both these inhibitors together completely blocks the oxidation of cytochrome b by quinones. In contrast, the simultaneous addition of antimycin and myxothiazol has no such synergistic effect upon the oxidation of cytochrome b by cytochrome c. Our data show that intramolecular electron transfer from cytochrome(s) b to the Rieske iron-sulphur center can take place in the bc1 complex without involvement of endogenous ubiquinone-10. This electron pathway is sensitive to all the inhibitors of the enzyme.     

Similar transition states mediate the Q-cycle and superoxide production by the cytochrome bc1 complex

The cytochrome bc complexes found in mitochondria, chloroplasts and many bacteria play critical roles in their respective electron transport chains. The quinol oxidase (Q(o)) site in this complex oxidizes a hydroquinone (quinol), reducing two one-electron carriers, a low potential cytochrome b heme and the "Rieske" iron-sulfur cluster. The overall electron transfer reactions are coupled to transmembrane translocation of protons via a "Q-cycle" mechanism, which generates proton motive force for ATP synthesis. Since semiquinone intermediates of quinol oxidation are generally highly reactive, one of the key questions in this field is: how does the Q(o) site oxidize quinol without the production of deleterious side reactions including superoxide production? We attempt to test three possible general models to account for this behavior: 1) The Q(o) site semiquinone (or quinol-imidazolate complex) is unstable and thus occurs at a very low steady-state concentration, limiting O(2) reduction; 2) the Q(o) site semiquinone is highly stabilized making it unreactive toward oxygen; and 3) the Q(o) site catalyzes a quantum mechanically coupled two-electron/two-proton transfer without a semiquinone intermediate. Enthalpies of activation were found to be almost identical between the uninhibited Q-cycle and superoxide production in the presence of antimycin A in wild type. This behavior was also preserved in a series of mutants with altered driving forces for quinol oxidation. Overall, the data support models where the rate-limiting step for both Q-cycle and superoxide production is essentially identical, consistent with model 1 but requiring modifications to models 2 and 3.