Reduction of cytochrome b-561 through the antimycin-sensitive site of the ubiquinol-cytochrome c2 oxidoreductase complex of Rhodopseudomonas sphaeroides

Cytochrome b-561 of the ubiquinol-cytochrome c2 oxidoreductase complex of Rhodopseudomonas sphaeroides is reduced after flash illumination in the presence of myxothiazol in an antimycin-sensitive reaction. Flash-induced reduction was observed over the redox range in which cytochrome b-561 and the Q-pool are both oxidized before the flash. The extent of reduction increased with increasing pH, and was maximal at pH greater than 10.0 where the extent approached that observed in the presence of antimycin following a group of flashes. Reduction of cytochrome b-561 in the presence of myxothiazol showed a lag of approximately 1 ms after the flash, followed by reduction with t 1/2 approximately 6 ms; by analogy with the similar kinetics of the quinol oxidase site, we suggest that the rate is determined by collision with the QH2 produced in the pool on flash excitation.     

Reversible inhibition of the mitochondrial ubiquinol-cytochrome c oxidoreductase complex (complex III) by ethoxyformic anhydride

The mitochondrial ubiquinol-cytochrome c oxidoreductase (complex III) is inhibited by ethoxyformic anhydride (EFA). The inhibition is readily reversed by hydroxylamine, suggesting the involvement of essential histidyl or possibly tyrosyl residues. The spectrum of ethoxyformylated complex III in the UV region showed a peak at 238 nm, indicative of N-(ethoxyformyl)histidine. Addition of hydroxylamine caused a large decrease of the 238-nm peak, which amounted to 16 mol of (ethoxyformyl)histidine/mol of cytochrome c1. Hydroxylamine addition to ethoxyformylated complex III also caused a small change at about 280 nm, which could be due to reversal of 1.6 O-ethoxyformylated tyrosyl residues/mol of cytochrome c1. Among many inhibitors of the cytochrome bc1 region of the respiratory chain, EFA is the only reagent known to cause reversible inhibition by covalent modification of amino acid residues. The inhibition site of EFA was determined to be between cytochromes b-562 and c1. However, unlike antimycin, which also inhibits in the same region, EFA did not promote the reduction of cytochrome b-566 in particles treated with substrates. In addition, it was found that EFA inhibits proton translocation in the cytochrome bc1 region and is a more effective electron transport inhibitor when added to reduced particles as compared to oxidized particles. These results together with the strong possibility that the EFA target is a histidyl or possibly a tyrosyl residue have been discussed in relation to the mechanism of proton translocation by complex III.     

Comparative biochemistry of the ubiquinol-cytochrome c oxidoreductase (EC 1.10.2.2) isolated from different heart mitochondria

The ubiquinol-cytochrome c oxidoreductase (bc1 complex, EC 1.10.2.2) has been isolated from the heart mitochondria of beef, chicken, turkey, duck and tuna with an identical procedure. The polypeptide composition of the different complexes, compared using SDS-polyacrylamide gel electrophoresis, shows that the three subunits carrying the prosthetic groups of the enzyme are highly conserved in all species. Also the large subunits I and II (core proteins) and band VI appear to be conserved in structure, while subunits VII and VIIa show a most remarkable structural variation in the various complexes. The steady-state ubiquinol-cytochrome c reductase analysis of the active enzymes indicates that all the bc1 complexes follow essentially a ping-pong mechanism, with the cytochrome c substrate displaying a partial competitive inhibition vs the ubiquinol substrate. The cytochrome c specificity of the reductase activity clearly is different in the various bc1 complexes, whereas the quinol specificity appears to be identical in all the enzymes.     

Mitochondrial myopathy involving ubiquinol-cytochrome c oxidoreductase (complex III) identified by immunoelectron microscopy

The distribution of respiratory chain complexes in bovine heart and human muscle mitochondria has been explored by immunoelectron microscopy with antibodies made against bovine heart mitochondrial proteins in conjunction with protein A-colloidal gold (12-nm particles). The antibodies used were made against NADH-coenzyme Q reductase (complex I), ubiquinol cytochrome c oxidoreductase (complex III), cytochrome c oxidase, core proteins isolated from complex III and the non-heme iron protein of complex III. Labeling of bovine heart tissue with any of these antibodies gave gold particles randomly distributed along the mitochondrial inner membrane. The labeling of muscle tissue from a patient with a mitochondrial myopathy localized by biochemical analysis to complex III was quantitated and compared with the labeling of human control muscle tissue. Complex I and cytochrome c oxidase antibodies reacted to the same level in myopathic and normal muscle samples. Antibodies to complex III or its components reacted very poorly to the patient's tissue but strongly to control muscle samples. Immunoelectron microscopy using respiratory chain antibodies appears to be a promising approach to the diagnosis and characterization of mitochondrial myopathies when only limited amounts of tissue are available for study.     

Mutants of ubiquinol-cytochrome c2 oxidoreductase resistant to Qo site inhibitors: consequences for ubiquinone and ubiquinol affinity and catalysis

Seven single-site mutants in six residues of the cyt b polypeptide of Rhodobacter capsulatus selected for resistance to the Qo site inhibitors stigmatellin, myxothiazol, or mucidin [Daldal, F., Tokito, M.K., Davidson, E., & Faham, M. (1989) EMBO J. 8, 3951-3961] have been characterized by using optical and EPR spectroscopy and single-turnover kinetic analysis. The strains were compared with wild-type strain MT1131 and with the Ps- strain R126 (G158D), which is dysfunctional in its Qo site [Robertson, D.E., Davidson, E., Prince, R.C., van den Berg, W.H., Marrs, B.L., & Dutton, P.L. (1986) J. Biol. Chem. 261, 584-591]. Mutants selected for stigmatellin resistance induced a weakening in the binding of the inhibitor without discernible loss of ubiquinone(Q)/ubiquinol(QH2) binding affinity to the Qo site or kinetic impairment to catalysis. Mutants selected for myxothiazol or mucidin resistance, inducing weakening of inhibitor binding, all displayed impaired rates of Qo site catalysis: The most severe cases (F144L, F144S) displayed loss of affinity for Q, and evidence suggests that parallel loss of affinity for the substrate QH2 was incurred in these strains. The results provide a view of the nature of the interaction of Q and QH2 of the Qpool with the Qo site. Consideration of the mutational substitutions and their structural positions along with comparisons with the QA and QB sites of the photosynthetic reaction center suggests a model for the structure of the Qo site.     

Decreased NADH dehydrogenase and ubiquinol-cytochrome c oxidoreductase in peripheral arterial disease

Peripheral arterial disease (PAD) is associated with muscle metabolic changes that may contribute to the disability in these patients. However, the biochemical defects in PAD have not been identified. The present study was undertaken to test the hypothesis that PAD is associated with specific defects in skeletal muscle electron transport chain activity. Seventeen patients with PAD and nine age-matched controls underwent gastrocnemius muscle biopsies. There were no differences in the mitochondrial content per gram of skeletal muscle as assessed by citrate synthase activity between the PAD patients and the control subjects. Skeletal muscle NADH dehydrogenase activity was decreased by 27% compared with controls when expressed per unit of citrate synthase activity. Expression of enzyme activities normalized to cytochrome c-oxygen oxidoreductase activity confirmed a 26% decrease in NADH dehydrogenase activity and also demonstrated a 38% decrease in ubiquinol-cytochrome c oxidoreductase activity. Thus PAD is associated with specific changes in muscle mitochondrial electron transport chain activities characterized by relative decreases in NADH dehydrogenase and ubiquinol-cytochrome c oxidoreductase activities, which may contribute to the metabolic abnormalities and decreased exercise performance in these patients.     

Modifications of redox equilibria with semiquinone stabilization upon pyruvate binding to L-lactate cytochrome c oxidoreductase (flavocytochrome b2)

Spectral redox titrations of flavin and cytochrome b₂ moieties of flavocytochrome b₂ were achieved in the absence and in the presence of pyruvate under equilibrium conditions at 18° C; direct measurements of spin flavosemiquinone proportions have been carried out by EPR determinations at the same temperature. Our results show that the equilibria involving flavin are largely affected by the presence of pyruvate; the semiquinone proportion markedly increases almost till unit near half-reduction of cytochrome b₂; at 10 mM pyruvate, the dismutation constant, $\text{K}{}_{\mathrm{dism}}\text{=}\text{(}\text{F}{}_{\mathrm{s}}\text{)}{}^2\text{(}\text{F}{}_{\mathrm{o}}\text{)}{}^1\text{(}\text{F}{}_{\mathrm{r}}\text{)}$ increases by a factor ≥ 10.     

Chromatographic and protein chemical analysis of the ubiquinol-cytochrome c2 oxidoreductase isolated from Rhodobacter sphaeroides

The ubiquinol-cytochrome c2 oxidoreductase (cytochrome bc1 complex) purified from chromatophores of Rhodobacter sphaeroides consists of four polypeptide subunits corresponding to cytochrome b, c1, and the Rieske iron-sulfur protein, as well as a 14-kDa polypeptide of unknown function, respectively. In contrast, the complex isolated from Rhodospirillum rubrum by the same procedure lacked a polypeptide corresponding to the 14-kDa subunit. Gel-permeation chromatography of the R. sphaeroides cytochrome bc1 complex in the presence of 200 mM NaCl removed the iron-sulfur protein, while the 14-kDa polypeptide remained tightly bound to the cytochromes; this is consistent with the possibility that the latter protein is an authentic component of the complex rather than an artifact of the isolation procedure. The individual polypeptides of the R. sphaeroides complex were purified to homogeneity by gel-permeation chromatography in the presence of 50% aqueous formic acid and their amino acid compositions determined. The 14-kDa polypeptide was found to be rich in charged and polar residues. Edman degradation analysis indicated that its N terminus is blocked and not rendered accessible by de-blocking procedures. Cyanogen bromide cleavage gave rise to a blocked N-terminal fragment as well as a C-terminal peptide comprising more than one-third of the protein. Gas-phase sequence analysis of this peptide established a sequence of 48 residues and identified a putative trans-membrane segment near the C terminus. The blocked N-terminal fragment was cleaved at tryptophan with BNPS-skatole. The resulting peptides, together with tryptic fragments derived from the intact protein, yielded additional sequence information; however, none of the sequences exhibited significant homologies to any known proteins. Tryptic fragments were also used to generate sequence information for cytochrome c1.     

Direct interaction between yeast NADH-ubiquinone oxidoreductase, succinate-ubiquinone oxidoreductase, and ubiquinol-cytochrome c oxidoreductase in the reduction of exogenous quinones

The reduction of the following exogenous quinones by succinate and NADH was studied in mitochondria isolated from both wild type and ubiquinone (Q)-deficient strains of yeast: ubiquinone-0 (Q0), ubiquinone-1 (Q1), ubiquinone-2 (Q2), and its decyl analogue 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), duroquinone (DQ), menadione (MQ), vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone), the plastoquinone analogue 2,3,6-trimethyl-1,4-benzoquinone (PQOc1), plastoquinone-2 (PQ2), and its decyl analogue (2,3-dimethyl-6-decyl-1,4-benzoquinone). Reduction of the small quinones DQ, Q0, Q1, and PQOc1 by NADH occurred in both wild type and Q-deficient mitochondria in a reaction inhibited more than 50% by myxothiazol and less than 20% by antimycin. The reduction of these small quinones by succinate also occurred in wild type mitochondria in a reaction inhibited more than 50% by antimycin but did not occur in Q-deficient mitochondria suggesting that endogenous Q6 is involved in their reduction. In addition, the inhibitory effects of antimycin and myxothiazol, specific inhibitors of the cytochrome b-c1 complex, on the reduction of these small quinones suggest the involvement of this complex in the electron transfer reaction. By contrast, the reduction of Q2 and DB by succinate was insensitive to inhibitors and by NADH was 20-30% inhibited by myxothiazol suggesting that these analogues are directly reduced by the primary dehydrogenases. The dependence of the sensitivity to the inhibitors on the substrate used suggests that succinate-ubiquinone oxidoreductase interacts specifically with center i (the antimycin-sensitive site) and NADH ubiquinone oxidoreductase preferentially with center o (the myxothiazol-sensitive site) of the cytochrome b-c1 complex. The NADH dehydrogenase involved in the myxothiazol-sensitive quinone reduction faces the matrix side of the inner membrane suggesting that center o may be localized within the membrane at a similar depth as center i.     

Spatial organization of redox active centers in the bovine heart ubiquinol-cytochrome c oxidoreductase

We have examined the spatial organization of the redox active centers in the Site II segment of the bovine heart respiratory chain by using reconstituted proteoliposomes of ubiquinol-cytochrome c oxidoreductase (Complex III or cytochrome bc1 complex) and EPR techniques. 1) Mutual spin-spin interactions between intrinsic redox active centers were detected. The spin relaxation of the Rieske iron-sulfur cluster was enhanced by the paramagnetic cytochrome c1 and b566 hemes but not by cytochrome b562. 2) Relative distances of the individual redox active centers to the P-side and N-side surfaces of the reconstituted Complex III proteoliposome were measured by our paramagnetic probe method (Blum, H., Bowyer, J. R., Cusanovich, M. A., Waring, A. J., and Ohnishi, T. (1983) Biochim. Biophys. Acta 748, 418-428). The cytochrome b562 heme was shown to be close to the middle of the phospholipid bilayer, while the Rieske iron-sulfur cluster and cytochrome b566 heme were assigned to be near the P-side surface level of the membrane. This probe method is a low resolution technique from the structural viewpoint; however, it can provide direct and reliable assignment of the topographical locations of redox active centers within the membrane. This is the first direct demonstration of the transmembranous location of the two cytochrome b hemes, although electron transfer between these two hemes crosses only half of the membrane thickness. Our data support the assignment of transmembranous distribution of the redox active centers based on electrochromic measurements (Robertson, D.E., and Dutton, P.L. (1988) Biochim, Biophys. Acta 935, 273-291). The implication of these results on the mechanism of Site II energy coupling is discussed.     

The effects of protein crowding in bacterial photosynthetic membranes on the flow of quinone redox species between the photochemical reaction center and the ubiquinol-cytochrome c2 oxidoreductase

Atomic force microscopy (AFM) of the native architecture of the intracytoplasmic membrane (ICM) of a variety of species of purple photosynthetic bacteria, obtained at submolecular resolution, shows a tightly packed arrangement of light harvesting (LH) and reaction center (RC) complexes. Since there are no unattributed structures or gaps with space sufficient for the cytochrome bc(1) or ATPase complexes, they are localized in membrane domains distinct from the flat regions imaged by AFM. This has generated a renewed interest in possible long-range pathways for lateral diffusion of UQ redox species that functionally link the RC and the bc(1) complexes. Recent proposals to account for UQ flow in the membrane bilayer are reviewed, along with new experimental evidence provided from an analysis of intrinsic near-IR fluorescence emission that has served to test these hypotheses. The results suggest that different mechanism of UQ flow exist between species such as Rhodobacter sphaeroides, with a highly organized arrangement of LH and RC complexes and fast RC electron transfer turnover, and Phaeospirillum molischianum with a more random organization and slower RC turnover. It is concluded that packing density of the peripheral LH2 antenna in the Rba. sphaeroides ICM imposes constraints that significantly slow the diffusion of UQ redox species between the RC and cytochrome bc(1) complex, while in Phs. molischianum, the crowding of the ICM with LH3 has little effect upon UQ diffusion. This supports the proposal that in this type of ICM, a network of RC-LH1 core complexes observed in AFM provides a pathway for long-range quinone diffusion that is unaffected by differences in LH complex composition or organization.     

A motif for quinone binding sites in respiratory and photosynthetic systems

Many of the membrane-bound protein complexes of respiratory and photosynthetic systems are reactive with quinones. To date, no clear structural relationship between sites that bind quinone has been defined, apart from that in the homologous family of "type II" photosynthetic reaction centres. We show here that a structural element containing a weak sequence motif is common to the Q(A) and Q(B) sites of bacterial reaction centres and the Q(i) site of the mitochondrial bc(1) complex. Analyses of sequence databases indicate that this element may also be present in the PsaA/B subunits of photosystem I, in the ND4 and ND5 subunits of complex I and, possibly, in the mitochondrial alternative quinol oxidase. This represents a first step in the structural classification of quinone binding sites.     

Demonstration of a collisional interaction of ubiquinol with the ubiquinol-cytochrome c2 oxidoreductase complex in chromatophores from Rhodobacter sphaeroides

Ubiquinone-10 can be extracted from lyophilized chromatophores of Rhodobacter sphaeroides (previously called Rhodopseudomonas sphaeroides) without significant losses in other components of the electron-transfer chain or irreversible damages in the membrane structure. The pool of ubiquinone can be restored with exogenous UQ-10 to sizes larger than the ones in unextracted membranes. The decrease in the pool size has marked effects on the kinetics of reduction of cytochrome b-561 induced by a single flash of light and measured in the presence of antimycin. The initial rate of reduction, which in unextracted preparations increases on reduction of the suspension over the Eh range between 170 and 100 mV (pH 7), is also stimulated in partially UQ-depleted membranes, although at more negative Eh's. When the UQ pool is completely extracted the rate of cytochrome (Cyt) b-561 reduction is low and unaffected by the redox potential. In membranes enriched in UQ-10 above the physiological level the titration curve of the rate of Cyt b-561 reduction is displaced to Eh values more positive than in controls. This effect is saturated when the size of the UQ pool is about 2-3 times larger than the native one. The reduction of Cyt b-561 always occurs a short time after the flash is fired; also the duration of this lag is dependent on Eh and on the size of the UQ pool. A decrease or an increase in the pool size causes a displacement of the titration curve of the lag to more negative or to more positive Eh's, respectively. Similarly, the lag becomes Eh independent and markedly longer than in controls when the pool is completely extracted. These results demonstrate that the rate of turnover of the ubiquinol oxidizing site in the b-c1 complex depends on the actual concentration of ubiquinol present in the membrane and that ubiquinol from the pool is oxidized at this site with a collisional mechanism. Kinetic analysis of the data indicates that this reaction obeys a Michaelis-Menten type equation, with a Km of 3-5 ubiquinol molecules per reaction center.     

Allosteric nucleotide-binding site in the mitochondrial NADH:ubiquinone oxidoreductase (respiratory complex I)

The rotenone-insensitive NADH:hexaammineruthenium III (HAR) oxidoreductase reactions catalyzed by bovine heart and Yarrowia lipolytica submitochondrial particles or purified bovine complex I are stimulated by ATP and other purine nucleotides. The soluble fraction of mammalian complex I (FP) and prokaryotic complex I homolog NDH-1 in Paracoccus denitrificans plasma membrane lack stimulation of their activities by ATP. The stimulation appears as a decrease in apparent K(m) values for NADH and HAR. Thus, the "accessory" subunits of eukaryotic complex I bear an allosteric ATP-binding site.     

Triple inhibitor titrations support the functionality of the dimeric character of mitochondrial ubiquinol-cytochrome c oxidoreductase.

The ubiquinol-2 or duroquinol oxidoreductase activity of mitochondrial ubiquinol-cytochrome c oxidoreductase was titrated with combinations of antimycin, myxothiazol and N,N'-dicyclohexylcarbodiimide (DCCD). A statistical model has been developed that can predict the activity of the complex treated with all possible combinations of these inhibitors. On the basis of the measured titration curves the model had to accommodate interaction between the two promoters of the complex. The titrations confirm that treatment with DCCD results in the modification of a certain site in one of the two promoters of the bc1 dimer, thereby blocking one antimycin A binding site without inhibiting electron transfer. Modification of both antimycin A binding sites of the dimer is apparently required for inhibition of electron transfer through the complex, just as modification of both myxothiazol-binding sites is required for full inhibition. The conclusion can be drawn that mitochondrial ubiquinol-cytochrome c oxidoreductase is a functional dimer, consisting of electrically interacting protomers.     

The existence of an antimycin A insensitive ubiquinol-cytochrome c reductase activity in the photosynthetic apparatus

A nonproteinaceous, antimycin A insensitive ubiquinol-cytochrome c reductase activity is detected in and purified from chromatophores of Rhodopseudomonas sphaeroides, R-26. This activity is about 5 times the antimycin A sensitive reductase activity in chromatophores and the two are not interconvertable. The purification involved chloroform:methanol (2:1), and hexane extractions and florisil column chromatography. The purified preparation contains some bacteriochlorophyll-like pigments and phospholipids, and is stable in organic solvent. It catalyzes the oxidation of ubiquinol by cytochrome c with substrate specificity and pH optimum.     

The inhibitory binding site(s) of Zn2+ in cytochrome c oxidase

EXAFS analysis of Zn binding site(s) in bovine-heart cytochrome c oxidase and characterization of the inhibitory effect of internal zinc on respiratory activity and proton pumping of the liposome reconstituted oxidase are presented. EXAFS identifies tetrahedral coordination site(s) for Zn(2+) with two N-histidine imidazoles, one N-histidine imidazol or N-lysine and one O-COOH (glutamate or aspartate), possibly located at the entry site of the proton conducting D pathway in the oxidase and involved in inhibition of the oxygen reduction catalysis and proton pumping by internally trapped zinc.     

Cytochrome electron spin resonance line shapes, ligand fields, and components stoichiometry in ubiquinol-cytochrome c oxidoreductase

The EPR spectra of the cytochromes in ubiquinol-cytochrome c oxidoreductase (Complex III) have peaks at g = 3.78 (cytochrome b566) g = 3.45 (cytochrome b562) and g = 3.35 (cytochrome c1). The highly asymmetric peak of cytochrome b566 has been simulated using an arbitrary gaussian distribution of crystal field parameters. The asymmetry is due to the nonlinear relationship between field position and crystal field. The results suggest that the b cytochromes have bis-imidazole ligation. The gz peak of cytochrome c1 was also found to be asymmetric; simulations suggest histidine-methionine ligation. No other important cytochrome components were needed to simulate the spectrum of the oxidized complex; these results are consistent with 1:1:1 stoichiometry of components. These results argue against any asymmetric dimer model for Complex III.     

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.