Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor

The cytochrome bc1 complex resides in the inner membrane of mitochondria and transfers electrons from ubiquinol to cytochrome c. This electron transfer is coupled to the translocation of protons across the membrane by the protonmotive Q cycle mechanism. This mechanism topographically separates reduction of quinone and reoxidation of quinol at sites on opposite sites of the membrane, referred to as center N (Qn site) and center P (Qp site), respectively. Both are located on cytochrome b, a transmembrane protein of the bc1 complex that is encoded on the mitochondrial genome. To better understand the parameters that affect ligand binding at the Qn site, we applied the Qn site inhibitor ilicicolin H to select for mutations conferring resistance in Saccharomyces cerevisiae. The screen resulted in seven different single amino acid substitutions in cytochrome b rendering the yeast resistant to the inhibitor. Six of the seven mutations have not been previously linked to inhibitor resistance. Ubiquinol-cytochrome c reductase activities of mitochondrial membranes isolated from the mutants confirmed that the differences in sensitivity toward ilicicolin H originated in the cytochrome bc1 complex. Comparative in vivo studies using the known Qn site inhibitors antimycin and funiculosin showed little cross-resistance, indicating different modes of binding of these inhibitors at center N of the bc1 complex.     

The dimeric structure of the cytochrome bc(1) complex prevents center P inhibition by reverse reactions at center N

Energy transduction in the cytochrome bc(1) complex is achieved by catalyzing opposite oxido-reduction reactions at two different quinone binding sites. We have determined the pre-steady state kinetics of cytochrome b and c(1) reduction at varying quinol/quinone ratios in the isolated yeast bc(1) complex to investigate the mechanisms that minimize inhibition of quinol oxidation at center P by reduction of the b(H) heme through center N. The faster rate of initial cytochrome b reduction as well as its lower sensitivity to quinone concentrations with respect to cytochrome c(1) reduction indicated that the b(H) hemes equilibrated with the quinone pool through center N before significant catalysis at center P occurred. The extent of this initial cytochrome b reduction corresponded to a level of b(H) heme reduction of 33%-55% depending on the quinol/quinone ratio. The extent of initial cytochrome c(1) reduction remained constant as long as the fast electron equilibration through center N reduced no more than 50% of the b(H) hemes. Using kinetic modeling, the resilience of center P catalysis to inhibition caused by partial pre-reduction of the b(H) hemes was explained using kinetics in terms of the dimeric structure of the bc(1) complex which allows electrons to equilibrate between monomers.     

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.     

Cytochrome bc1 complexes of microorganisms.

The cytochrome bc1 complex is the most widely occurring electron transfer complex capable of energy transduction. Cytochrome bc1 complexes are found in the plasma membranes of phylogenetically diverse photosynthetic and respiring bacteria, and in the inner mitochondrial membrane of all eucaryotic cells. In all of these species the bc1 complex transfers electrons from a low-potential quinol to a higher-potential c-type cytochrome and links this electron transfer to proton translocation. Most bacteria also possess alternative pathways of quinol oxidation capable of circumventing the bc1 complex, but these pathways generally lack the energy-transducing, protontranslocating activity of the bc1 complex. All cytochrome bc1 complexes contain three electron transfer proteins which contain four redox prosthetic groups. These are cytochrome b, which contains two b heme groups that differ in their optical and thermodynamic properties; cytochrome c1, which contains a covalently bound c-type heme; and a 2Fe-2S iron-sulfur protein. The mechanism which links proton translocation to electron transfer through these proteins is the proton motive Q cycle, and this mechanism appears to be universal to all bc1 complexes. Experimentation is currently focused on understanding selected structure-function relationships prerequisite for these redox proteins to participate in the Q-cycle mechanism. The cytochrome bc1 complexes of mitochondria differ from those of bacteria, in that the former contain six to eight supernumerary polypeptides, in addition to the three redox proteins common to bacteria and mitochondria. These extra polypeptides are encoded in the nucleus and do not contain redox prosthetic groups. The functions of the supernumerary polypeptides of the mitochondrial bc1 complexes are generally not known and are being actively explored by genetically manipulating these proteins in Saccharomyces cerevisiae.     

Structural basis for the mechanism of electron bifurcation at the quinol oxidation site of the cytochrome <Emphasis Type="Italic">bc</Emphasis><Subscript>1</Subscript> complex

At the heart of the Q cycle hypothesis, the cytochrome bc1 complex (bc1) is required to separate the two electrons from a quinol molecule at the quinol oxidation site. Recent studies have brought to light an intricate mechanism for this bifurcated electron transfer. A survey of the protein data bank shows 30 entries for the structures of bc1 and the homologous b6 f complex. These structures provide considerable insights into the structural organization of mitochondrial, bacterial, and plant enzymes. Crystallographic binding studies of bc1 with either quinone reduction (QN) and/or quinol oxidation (QP) site inhibitors offer atomic details on how these compounds interact with residues at their respective sites. Most importantly, the different locations and apparent flexibility observed in crystals for the extrinsic domain of the iron-sulfur protein (ISP) subunit suggest a mechanism for electron bifurcation at the QP site. Analyses of various inhibitor-bound structures revealed two classes of QP site inhibitors: Pm inhibitors that promote ISP mobility and Pf inhibitors that favor the fixation of the ISP conformation. Those analyses also shed light on a possible process by which the ISP motion switch is controlled. The first phase reduction of ISP is shown to be comparable to the reduction of the bL heme by pre-steady state kinetic analysis, whereas the second phase reduction of ISP share similar kinetics with the reduction of the bH heme. The reduction of cyt c1 is measured much slower, indicating that the reduced ISP remains bound at the QP site until the reduced heme bL is oxidized by the heme bH and supporting the existence of a control mechanism for the ISP motion switch.     

Myxothiazol resistance in human mitochondria

We have investigated electron transfer activities of respiratory chain complexes in platelet mitochondria of a patient with intermittent ataxia and lactic acidosis who was previously reported to be deficient in the E1 (decarboxylase) component of the pyruvate dehydrogenase complex. Electron transfer from succinate to cytochrome c was normal, but the mitochondria exhibited moderately decreased (63% of control) quinol: cytochrome-c oxidoreductase activity, suggesting a defect in complex III. Consistent with some perturbation in complex III, electron flux through complex III was resistant to inhibition by myxothiazol compared to normal controls. In contrast, titration with antimycin revealed a less abnormal pattern of inhibition. The extreme specificity of myxothiazol binding at or near the quinol oxidase domain of mitochondrial cytochrome b, i.e., b-566, suggests a defect in this region of complex III which may perturb the kinetics or thermodynamics of quinol oxidation in the complex. These data suggest that the patient's illness results from a mutation in the quinol oxidase domain of mitochondrial cytochrome b (b-566).     

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.     

Inhibition of the yeast cytochrome bc1 complex by ilicicolin H, a novel inhibitor that acts at the Qn site of the bc1 complex

Ilicicolin H is an antibiotic isolated from the "imperfect" fungus Cylindrocladium iliciola strain MFC-870. Ilicicolin inhibits mitochondrial respiration by inhibiting the cytochrome bc(1) complex. In order to identify the site of ilicicolin action within the bc(1) complex we have characterized the effects of ilicicolin on the cytochrome bc(1) complex of Saccharomyces cerevisiae. Ilicicolin inhibits ubiquinol-cytochrome c reductase activity of the yeast bc(1) complex with an IC(50) of 3-5 nM, while 200-250 nM ilicicolin was required to obtain comparable inhibition of the bovine bc(1) complex. Ilicicolin blocks oxidation-reduction of cytochrome b through center N of the bc(1) complex and promotes oxidant-induced reduction of cytochrome b but has no effect on oxidation of ubiquinol through center P. These results indicate that ilicicolin binds to the Qn site of the bc(1) complex. Ilicicolin induces a blue shift in the absorption spectrum of ferro-cytochrome b, and titration of the spectral shift indicates binding of one inhibitor molecule per Qn site. The effects of ilicicolin on electron transfer reactions in the bc(1) complex are similar to those of antimycin, another inhibitor that binds to the Qn site of the bc(1) complex. However, because the two inhibitors have different effects on the absorption spectrum of cytochrome b, they differ in their mode of binding to the Qn site.     

Movement of the Rieske iron-sulfur protein in the p-side bulk aqueous phase: effect of lumenal viscosity on redox reactions of the cytochrome b6f complex

Based on the atomic structures of the mitochondrial cytochrome bc(1) complex, it has been proposed that the soluble domain of the [2Fe-2S] Rieske iron-sulfur protein (ISP) must rotate by ca. 60 degrees and translate through an appreciable distance between two binding sites, proximal to cytochrome c(1) and to the lumen-side quinol binding site. Such motional freedom implies that the electron-transfer rate should be affected by the lumenal viscosity. The flash-induced oxidation of cytochrome f, the chloroplast analogue of cytochrome c(1), was found to be inhibited reversibly by increased lumenal viscosity, as was the subsequent reduction of both cytochrome b(6) and cytochrome f. The rates of these three redox reactions correlated inversely with lumenal viscosity over a viscosity range of 1-10 cP. Reduction of cytochrome b(6) and cytochrome f was not concerted. The rate of cytochrome f reduction was observed to be approximately half that of cytochrome b(6) regardless of the actual viscosity, implying that the path length traversed by the ISP in reduction of cytochrome f is twice that of cytochrome b(6). This suggests that upon initiation of electron transfer by a light flash, cytochrome b(6) reduction requires movement of reduced ISP from an initial position predominantly proximal to cytochrome f, apparently favored by the reduced ISP, to the quinol binding site at which the oxidant-induced reduction of cytochrome b(6) is initiated. Subsequent reduction of cytochrome f requires the additional movement of the ISP back to a site proximal to cytochromef. There is no discernible viscosity dependence for cytochrome b(6) reduction under oxidizing conditions, presumably because the oxidized ISP preferentially binds proximal to the quinone binding niche. The dependence of the cytochrome redox reaction on ambient viscosity implies that the tethered diffusional motion of the ISP is part of the rate limitation for charge transfer through the b(6)f complex.     

Altered Mitochondrial Respiration in a Chromosomal Mutant of Neurospora crassa

A mutant of Neurospora crassa (cni-1) has been isolated that has two pathways of mitochondrial respiration. One pathway is sensitive to cyanide and antimycin A, the other is sensitive only to salicyl hydroxamic acid. Respiration can proceed through either pathway and both pathways together in this mutant account for greater than 90% of all mitochondrial respiration. The cni-1 mutation segregates as a nuclear gene in crosses to other strains of Neurospora. Absorption spectra of isolated mitochondria from cni-1 show typical b- and c-type cytochromes but the absorption peaks corresponding to cytochrome aa(3) are not detectable. Extraction of soluble cytochrome c-546 from these mitochondria followed by reduction with ascorbate reveals a new absorption peak at 426 nm that is not present in wild-type mitochondria. This peak may be due to an altered cytochrome oxidase with abnormal spectral properties. Mitochondria from cni-1 have elevated levels of succinate-cytochrome c reductase but reduced levels of nicotinamide adenine dinucleotide reduced form cytochrome c reductase and of cyanide- and azide-sensitive cytochrome c oxidase. These studies suggest that the cni-1 mutation results in the abnormal assembly of cytochrome c oxidase so that the typical cytochrome aa(3) spectrum is lost and the enzyme activity is reduced. As a consequence of this alteration, a cyanide-insensitive respiratory pathway is elaborated by these mitochondria which may serve to stimulate adenosine 5'-triphosphate production via substrate level phosphorylation by glycolysis and the Krebs cycle.     

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.     

An Engineered Cytochrome b6c1 Complex with a Split Cytochrome b Is Able To Support Photosynthetic Growth of Rhodobacter capsulatus

The ubihydroquinone-cytochrome c oxidoreductase (or the cytochrome bc1 complex) from Rhodobacter capsulatus is composed of the Fe-S protein, cytochrome b, and cytochrome c1 subunits encoded by petA(fbcF), petB(fbcB), and petC(fbcC) genes organized as an operon. In the work reported here, petB(fbcB) was split genetically into two cistrons, petB6 and petBIV, which encoded two polypeptides corresponding to the four amino-terminal and four carboxyl-terminal transmembrane helices of cytochrome b, respectively. These polypeptides resembled the cytochrome b6 and su IV subunits of chloroplast cytochrome b6f complexes, and together with the unmodified subunits of the cytochrome bc1 complex, they formed a novel enzyme, named cytochrome b6c1 complex. This membrane-bound multisubunit complex was functional, and despite its smaller amount, it was able to support the photosynthetic growth of R. capsulatus. Upon further mutagenesis, a mutant overproducing it, due to a C-to-T transition at the second base of the second codon of petBIV, was obtained. Biochemical analyses, including electron paramagnetic spectroscopy, with this mutant revealed that the properties of the cytochrome b6c1 complex were similar to those of the cytochrome bc1 complex. In particular, it was highly sensitive to inhibitors of the cytochrome bc1 complex, including antimycin A, and the redox properties of its b- and c-type heme prosthetic groups were unchanged. However, the optical absorption spectrum of its cytochrome bL heme was modified in a way reminiscent of that of a cytochrome b6f complex. Based on the work described here and that with Rhodobacter sphaeroides (R. Kuras, M. Guergova-Kuras, and A. R. Crofts, Biochemistry 37:16280-16288, 1998), it appears that neither the inhibitor resistance nor the redox potential differences observed between the bacterial (or mitochondrial) cytochrome bc1 complexes and the chloroplast cytochrome b6f complexes are direct consequences of splitting cytochrome b into two separate polypeptides. The overall findings also illustrate the possible evolutionary relationships among various cytochrome bc oxidoreductases.     

Cytochrome c1 of bakers' yeast. II. Synthesis on cytoplasmic robosomes and influence of oxygen and heme on accumulation of the apoprotein.

In the preceding paper (Ross, E., and Schatz, G. (1976) J. Biol. Chem. 251, 1991-1996) yeast cytochrome c1 was characterized as a 31,000 dalton polypeptide with a covalently bound heme group. In order to determine the site of translation of this heme-carrying polypeptide, yeast cells were labeled with [H]leu(be under the following conditions: (a) in the absence of inhibitors, (b) in the presence of acriflavin (an inhibitor of mitochondrial translation), or (c) in the presence of cycloheximide (an inhibitor of cytoplasmic translation). The incorporation of radioactivity into the hemeprotein was measured by immunoprecipitating it from mitochondrial extracts and analyzing it by dodecyl sulfate-polyacrylamide gel electrophoresis. Label was incorporated into the cytochrome c1 apoprotein only in the presence of acriflavin or in the absence of inhibitor, but not in the presence of cycloheximide. Cytochrome c1 is thus a cytoplasmic translation product. This conclusion was further supported by the demonstration that a cytolasmic petite mutant lacking mitochondrial protein synthesis still contained holocytochrome c1 that was indistinguishable from cytochrome c1 of wild type yeast with respect to molecular weight, absorption spectru, the presence of a covalently bound heme group, and antigenic properties. Cytochrome c1 in the mitochondria of the cytoplasmic petite mutant is firmly bound to the membrane, and its concentration approaches that typical of wild type mitochondria. However, its lability to proteolysis appeared to be increased. A mitochondrial translation product may thus be necessary for the correct conformation or orientation of cytochrome c1 in the mitochondrial inner membrane. Accumulation of cytochrome c1 protein in mitochondria is dependent on the abailability of heme. This was shown with a delta-aminolevulinic acid synthetase-deficient yeast mutant which lacks heme and any light-absorbing peaks attributable to cytochromes. Mitochondria from mutant cells grown without added delta-aminolevulinic acid contained at least 20 times less protein immunoprecipitable by cytochrome c1-antisera than mitochondria from cells grown in the presence of the heme precursor. Similarly, the respiration-deficient promitochondria of anaerobically grown wild type cells are almost completely devoid of material cross-reacting with cytochrome c1-antisera. A 105,000 X g supernatant of aerobically grown wild type cells contains a 29,000 dalton polypeptide that is precipitated by cytochrome c1-antiserum but not by nonimmune serum. This polypeptide is also present in high speed supernatants from the heme-deficient mutant or from anaerobically gorwn wild type cells. The possible identity of this polypeptide with soluble apocytochrome c1 is being investigated.     

Identification and properties of a quinol oxidase super-complex composed of a bc1 complex and cytochrome oxidase in the thermophilic bacterium PS3

Evidence for the presence of a quinol oxidase super-complex composed of a cytochrome bc1 complex and cytochrome oxidase in the respiratory chain of a Gram-positive thermophilic bacterium PS3 is reported. On incubation with an octyl glucoside-solubilized fraction of the total membranes of PS3 anti-serum against PS3 cytochrome oxidase gave an immunoprecipitate that showed both quinol-cytochrome c reductase and cytochrome c oxidase activities. When the cholate-deoxycholate and LiCl-treated membranes of PS3 were solubilized and subjected to ion-exchange chromatography in the presence of octaethyleneglycol dodecyl ether, most of the A-, B-, and C-type cytochromes were copurified as a peak having both quinol-cytochrome c reductase and cytochrome oxidase activities. The immunoprecipitate and quinol oxidase preparation contained hemes a, b, and c in a ratio of about 2:2:3, indicating the presence of one-to-one complex of cytochrome oxidase containing 2 hemes a and one heme c, and a bc1 complex containing 2 hemes b and 2 hemes c. Gel electrophoresis in the presence of dodecyl sulfate showed that the immunoprecipitate and quinol oxidase preparation were composed of seven subunits; those of 51 (56-kDa), 38, and 22 kDa for cytochrome oxidase and those of 29, 23, 21, and 14 kDa for the bc1 complex. The 38-, 29-, and 21 kDa components possessed covalently bound heme c. The apparent molecular mass of the super complex was estimated to be as 380 kDa by gel filtration.     

Stimulation of the respiratory chain of rat liver mitochondria between cytochrome c1 and cytochrome c by glucagon treatment of rats

Mitochondria from glucagon-treated rats oxidize succinate, but not ascorbate plus tetramethylphenylenediamine, faster in the uncoupled state than do control mitochondria. The rate of O(2) uptake in the presence of both substrates is equal to the sum of the rates of the O(2) uptake in the presence of either substrate alone. It is concluded that the mitochondrial respiratory chain is limited at some point between cytochromes b and c and that this step is regulated by glucagon. Measurement of the cytochrome spectra under uncoupled conditions in the presence of succinate and rotenone demonstrates a crossover between cytochromes c and c(1) when control mitochondria are compared with those from glucagon-treated rats, cytochrome c being more oxidized and cytochrome c(1) more reduced in control mitochondria. Under conditions where pyruvate metabolism is studied the control mitochondria are generally more oxidized than those from glucagon-treated rats, the redox state of cytochrome b-566 correlating with the rate of pyruvate metabolism in sucrose medium. However, when the redox state of the mitochondria is taken into account, a crossover between cytochromes c and c(1) is again apparent. The spectra of the b cytochromes are complex, but cytochrome b-562 appears to become more reduced relative to cytochrome b-566 in mitochondria from glucagon-treated rats than in control mitochondria. This can be explained by the existence of a more alkaline matrix in glucagon-treated rats, the redox potential for cytochrome b being pH-sensitive. It is concluded that glucagon stimulates electron flow between cytochromes c(1) and c. The physiological significance of these findings is discussed.     

Protonmotive pathways and mechanisms in the cytochrome bc1 complex

The cytochrome bc(1) complex catalyzes electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane. In the Q cycle mechanism proton translocation is the net result of topographically segregated reduction of quinone and reoxidation of quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the quinol. The linkage of proton chemistry to electron transfer during quinol oxidation and quinone reduction requires pathways for moving protons to and from the aqueous phase and the hydrophobic environment in which the quinol and quinone redox reactions occur. Crystal structures of the mitochondrial cytochrome bc(1) complexes in various conformations allow insight into possible proton conduction pathways. In this review we discuss pathways for proton conduction linked to ubiquinone redox reactions with particular reference to recently determined structures of the yeast bc(1) complex.     

The cytochrome ba complex from the thermoacidophilic crenarchaeote Acidianus ambivalens is an analog of bc(1) complexes

A novel cytochrome ba complex was isolated from aerobically grown cells of the thermoacidophilic archaeon Acidianus ambivalens. The complex was purified with two subunits, which are encoded by the cbsA and soxN genes. These genes are part of the pentacistronic cbsAB-soxLN-odsN locus. The spectroscopic characterization revealed the presence of three low-spin hemes, two of the b and one of the a(s)-type with reduction potentials of +200, +400 and +160 mV, respectively. The SoxN protein is proposed to harbor the heme b of lower reduction potential and the heme a(s), and CbsA the other heme b. The soxL gene encodes a Rieske protein, which was expressed in E. coli; its reduction potential was determined to be +320 mV. Topology predictions showed that SoxN, CbsB and CbsA should contain 12, 9 and one transmembrane alpha-helices, respectively, with SoxN having a predicted fold very similar to those of the cytochromes b in bc(1) complexes. The presence of two quinol binding motifs was also predicted in SoxN. Based on these findings, we propose that the A. ambivalens cytochrome ba complex is analogous to the bc(1) complexes of bacteria and mitochondria, however with distinct subunits and heme types.     

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.     

Reaction of Escherichia coli cytochrome bo(3) and mitochondrial cytochrome bc(1) with a photoreleasable decylubiquinol

In order to probe the reaction chemistry of respiratory quinol-oxidizing enzymes on a rapid time scale, a photoreleasable quinol substrate was synthesized by coupling decylubiquinol with the water-soluble protecting group 3',5'-bis(carboxymethoxy)benzoin (BCMB) through a carbonate linkage. The resulting compound, DQ-BCMB, was highly soluble in aqueous detergent solution, and showed no reactivity with quinol-oxidizing enzymes prior to photolysis. Upon photolysis in acetonitrile, 5, 7-bis(carboxymethoxy)-2-phenylbenzofuran, carbon dioxide, and decylubiquinol were formed. In aqueous media, free 3', 5'-bis(carboxymethoxy)benzoin was also produced. Photolysis of DQ-BCMB with a 308 nm excimer laser led to the release of the BCMB group in less than 10(-6) s. Decylubiquinol was released in the form of a carbonate monoester, which decarboxylated with an observed first-order rate constant of 195-990 s(-1), depending on the reaction medium. Yields of decylubiquinol as high as 35 microM per laser pulse were attained readily. In the presence of Escherichia coli cytochrome bo(3), photolysis of DQ-BCMB led to the oxidation of quinol by the enzyme with a rate that was limited by the rate of the decylubiquinol release. Mitochondrial cytochrome bc(1) reacted with photoreleased decylubiquinol with distinct kinetic phases corresponding to rapid b heme reduction and somewhat slower c heme reduction. Oxidation of photoreleased ubiquinol by this enzyme showed saturation kinetics with a K(m) of 3.6 microM and a k(cat) of 210 s(-1). The saturation behavior was a result of decylubiquinol being released as a carbonate monoester during the photolysis of DQ-BCMB and interacting with cytochrome bc(1) before decarboxylation of this intermediate yielded free decylubiquinol. The reaction of cytochrome bc(1) and photoreleased decylubiquinol in the presence of antimycin A led to monophasic b heme reduction, but also yielded slower quinol oxidation kinetics. The discrimination of kinetic phases in the reaction of cytochrome bc(1) with ubiquinol substrates has provided a means of exploring the bifurcation of electron transfer that is central to the operation of the Q-cycle in this enzyme.     

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.