Membrane topology of beef-heart ubiquinone-cytochrome c reductase (complex III)

The membrane topology of ubiquinone-cytochrome c reductase (EC 1.10.2.2.) has been investigated with photoreactive lipid analogs (Bisson, R., and Montecucco, C. (1981) Biochem. J. 193, 757-763), both in its isolated form and when part of succinate-cytochrome c reductase (Complex II + III). These probes react specifically with those polypeptide chains exposed to lipids, thereby labeling them radioactively. Highly resolving gel electrophoretic conditions have been used to determine the patterns of labeling. Core protein I, cytochrome b, cytochrome c1, and polypeptides VI, VII, VIII, and IX contribute to the lipid-protein boundary of Complex III. Evidence that the interaction between Complex II and Complex III involves their hydrophobic domains is also presented.     

Cross-linking of ubiquinone cytochrome c reductase (complex III) with periodate-cleavable bifunctional reagents.

Two novel cross-linkers, disuccinimidyl tartarate (DST) and N,N'-bis(3-succinimidyloxycarbonylpropyl)tartaramide (SPT), have been synthesized. These reagents span 6 and 18 A, respectively, between functional groups and contain a vic-glycol bond which can be cleaved with periodate under mild reaction conditions. Both DST and SPT have been used to examine the near-neighbor relationships of polypeptides in ubiquinone cytochrome c reductase (complex III) from beef heart mitochondria. Among the cross-linked products resolved were pairs containing I + II, II + VI, I + V, and VI + VII. Polypeptides III and IV, a cytochrome b aproprotein, and the cytochrome c1 hemoprotein, respectively, were also resolved in several cross-linked products.     

The polypeptide composition of ubiquinone-cytochrome c reductase (complex III) from beef heart mitochondria.

The subunit structure of ubiquinone-cytochrome c reductase (complex III) has been examined and eight different polypeptides have been identified. Apparent molecular weights for each have been obtained by one or more methods including polyacrylamide gel electrophoresis in sodium doecyl sulfate and in sodium dodecyl sulfate-8 M urea and by gel filtration in sodium dodecyl sulfate and in 6 M guanidine hydrochloride. Values obtained are as follows: I, 47 500; II, 45 500; III, 29 500; IV, 27 800; V, 24 800; VI, 13 900; VII, 10 700; VIII, 4 800-9 00. Individual polypeptides have been purified and the amino acid composition of several of these have been determined. At least one polypeptide, the apoprotein of cytochrome b, is hydrophobic in character and this is a mitochondrially synthesized component (B. Lorenz, W. Kleinow, and H. Weiss (1974), Hoppe-Seyler's Z. Physiol. Chem. 355, 300). Other polypeptides including the hemoprotein of cytochrome c1 are more hydrophilic in amino acid composition.     

Energized cation transport by complex III (ubiquinone-cytochrome C reductase)

Energized cyclical and net transport of cations and anions has been demonstrated in liposomes containing Complex III using a protamine-aggregation technique. Energization with reduced ubiquinone induces a rapid ion uptake followed by a more gradual efflux upon exhaustion of the available reductant. Both monovalent and divalent cations are transported, but at relatively high concentrations divalent cations are transported in preference to monovalent cations. Results are consistent with a cation:electron ratio of unity.     

Nearest neighbor relationships of the polypeptides in ubiquinone cytochrome c reductase (complex III).

Ubiquinone cytochrome c reductase (complex III) in detergent dispersion has been cross-linked with two reversible cross-linking agents dithiobissuccinimidylpropionate and dimethyl-3.3'-dithiobispropionimidate and the cross-linked products formed have been analyzed by two-dimensional gel electrophoresis. Under mild reaction conditions, polypeptides I and II, II and VI, I and V, and VI and VII were the most prominent subunit pairs seen. With higher levels of reagent, larger aggregates were produced until an aggregate of apparent molecular weight 310 000 was the dominant band on gels. This is the complex III monomer.     

Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria.

Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O₂^? at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H₂O₂ at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H₂O₂ was essentially produced by disproportionation of O₂^?, which constitutes the precursor of H₂O₂. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O₂^? and 0.63 mol of H₂O₂/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O₂ to O₂^?, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H₂O₂ generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H₂O₂ generation. The efficiency of added quinones as peroxide generators decreased in the order Q₁ > Q₀ > Q₂ > Q₆ = Q₁₀, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymatically by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O₂^? and H₂O₂ during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q₀H₂), benzoquinol, duroquinol and menadiol generated O₂^? with k₃ values of 0.1 to 1.4 m^? · s^?1 and H₂O₂ with k₄ values of 0.009 to 4.3 m^?1 · s^?1.     

A cytochrome b/c1 complex with ubiquinol--cytochrome c2 oxidoreductase activity from Rhodopseudomonas sphaeroides GA

A cytochrome b/c1 complex which catalyses the reduction of cytochrome c by ubiquinol has been isolated from Rhodopseudomonas sphaeroides GA. It contains two hemes b and substoichiometric amounts of ubiquinone-10 and of the Rieske Fe-S center per cytochrome c1, and is essentially free of reaction center and bacteriochlorophyll. The complex consists of three major polypeptides with apparent molecular masses of 40, 34 and 25 kDa. The 34-kDa polypeptide carries heme. Cytochrome c1 has a midpoint potential of 285 mV. For cytochrome b two midpoint potentials, at 50 and -60 mV, at pH 7.4, can be derived if one assumes two components of equal amount. Ubiquinol--cytochrome c oxidoreductase activity is specific for ubiquinol and bacterial cytochromes c, and is inhibited by antimycin A and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. The complex shows oxidant-induced reduction of cytochrome b.     

Immunological studies on the participation of 6-pyruvoyl tetrahydropterin (2'-oxo) reductase, an aldose reductase, in tetrahydrobiopterin biosynthesis

The NADPH-dependent reduction of the two carbonyl groups in the side chain of the first tetrahydropterin intermediate on the tetrahydrobiopterin biosynthetic pathway, 6-pyruvoyl tetrahydropterin, proceeds in a sequential manner whose order has not yet been resolved. Sepiapterin reductase can catalyze the reduction of both carbonyl groups starting with the 1'-oxo. 6-Pyruvoyl tetrahydropterin (2'-oxo) reductase, which has now been shown to be a member of the aldose reductase family, catalyzes the formation of only the 2'-hydroxy-1'-oxo intermediate which still requires sepiapterin reductase for final conversion to tetrahydrobiopterin. Inhibiting antibodies to the 2'-oxo reductase have been prepared and utilized to explore the distribution of this reductase in rat brain. The antiserum also maximally inhibited in vitro tetrahydrobiopterin synthesis in crude rat brain extracts by 60%, indicating that the majority of tetrahydrobiopterin biosynthesis in vivo may proceed via the 2'-hydroxy-1'-oxo intermediate. However, analogous experiments with rat liver extracts demonstrate that inhibition of the 2'-oxo reductase activity does not inhibit the conversion of 6-pyruvoyl tetrahydropterin to tetrahydrobiopterin, suggesting that tetrahydrobiopterin biosynthesis may proceed via different pathways in rat brain and liver.     

Human mitochondrial thioredoxin reductase reduces cytochrome c and confers resistance to complex III inhibition

The ubiquitously expressed mammalian thioredoxin reductases are selenoproteins that together with NADPH regenerate active reduced thioredoxins and are involved in diverse actions mediated by redox control. Two main forms of mammalian thioredoxin reductases have been isolated, one cytosolic (TrxR1) and one present in mitochondria (TrxR2). Although the principal target for TrxRs is thioredoxin, the cytosolic form can regenerate several important antioxidants such as ascorbic acid, lipoic acid, and ubiquinone. In this study we demonstrate that cytochrome c is a substrate for both TrxR1 and TrxR2. In addition, cells overexpressing TrxR2 are more resistant to impairment of complex III in the mitochondrial respiratory chain upon both antimycin A and myxothiazol treatments, suggesting a complex III bypassing function of TrxR2. Furthermore, we show that cytochrome c is reduced by TrxR2 in vitro, not only by using NADPH as an electron donor but also by using NADH, pointing at TrxR2 as an important redox protein on complex III impairment. These findings may be valuable in understanding respiratory disorders in mitochondrial diseases.     

Evidence for a function of core protein in complex III from beef-heart mitochondria.

Puried complex III ) ubiquinol-cytochrome c reductase) from beef heart mitochondria was alkylated with iodol [1-14C]acetamide. After 6-8 h of incubation with iodo[1-14C]acetamide, duroquinol and ubiquinol-2-cytochrome c reductase activites were inhibited approximately 50%. During this time 4.5 +/- 1.6 nmol of iodo[1-14C]acetamide reacted per mg of complex III protein. Experiments carried out over 24 h indicated that enzyme activity could be inhibited to 70% and the alkylation of complex III was proportional to inhibition. The rates of cytochrome b and c1 reduction by duroquinol are also decreased upon treatment of complex III with iodoacetamide. Separation of the peptides of complex III by electrophoresis in sodium dodecylsulfate shows that all of the radioactivity is located in a single peptide of 50 000 molecular weight, which has been identified as one of the two core proteins. The possible functions of core protein are discussed.     

Inhibition and inactivation of NADH-cytochrome c reductase activity of bovine heart submitochondrial particles by the iron(III)-adriamycin complex.

The NADH-cytochrome c reductase activity of bovine heart submitochondrial particles was found to be slowly (half-time of 16 min) and progressively lost upon incubation with the Fe2(+)-adriamycin complex. In addition to this slow progressive inactivation seen on incubation, a reversible fast phase of inhibition was also seen. However, if EDTA was added to the incubation mixture within 15 s, the slow progressive loss in activity was largely preventable. Separate experiments indicated that EDTA removed about one-half of the iron from the Fe2(+)-adriamycin complex in about 40 s. These results indicated the requirement for iron for the inactivation process. Since the Vmax. for the fast phase of inhibition was decreased by the inhibitor, the inhibition pattern was similar to that seen for uncompetitive or mixed-type inhibition. The direct binding of both Fe3(+)-adriamycin and adriamycin to submitochondrial particles was also demonstrated, with the Fe3(+)-adriamycin complex binding 8 times more strongly than adriamycin. Thus binding of Fe3(+)-adriamycin to the enzyme or to the inner mitochondrial membrane with subsequent generation of oxy radicals in situ is a possible mechanism for the Fe3(+)-adriamycin-induced inactivation of respiratory enzyme activity.     

A mitochondrial cytochrome b mutation but no mutations of nuclearly encoded subunits in ubiquinol cytochrome c reductase (complex III) deficiency

Ubiquinol cytochrome c reductase (complex III) deficiency represents a clinically heterogeneous group of mitochondrial respiratory chain disorders that can theoretically be subject to either a nuclear or a mitochondrial mode of inheritance. In an attempt to elucidate the molecular bases of the disease, we first determined the nucleotide sequence of three unknown subunits (9.5 kDa, 7.2 kDa, 6.4 kDa) by cyberscreening of human expressed sequence tag data bases and sequenced the 11 cDNA subunits encoding complex III in five patients with isolated complex III deficiency. No mutation in the nuclearly encoded complex III subunits was observed, but a mutation in the cd2 helix of the mitochondrial (mt) cytochrome b gene was found to alter the conformation of the bc ₁ complex in one patient with severe hypertrophic cardiomyopathy. The present study is highly relevant to genetic counseling as the absence of mtDNA mutations in all but one patient in our series strongly supports autosomal rather than maternal inheritance in the majority of patients with complex III deficiency. Received: 15 January 1999 / Accepted: 31 March 1999     

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.     

Analysis of the peptide composition of purified beef-heart complex III by dodecylsulfate electrophoresis.

The peptides of purified complex III from beef heart mitochondria have been studied by electrophoresis on dodecylsulfate gels. Of the 12 peptides consistently observed, only eight appear to be integral peptides of the functional complex. Attempts to identify these peptides have been made through co-electrophoresis of complex III and fractions in which the individual peptides were either purified or greatly enriched. Electrophoresis of complex III preparations which were not reduced by mercaptoethanol indicates that intermolecular disulfide bonds play no significant role in stabilizing the complex.     

Potentiometric studies on yeast complex III

Potentiometric measurements have been performed on Complex III from bakers' yeast. The midpoint potentials for the b and c cytochromes were measured using room-temperature MCD and liquid-helium temperature EPR. A value of 270 mV was obtained for cytochrome c1, regardless of temperature, while the midpoint potentials found for the two species of cytochrome b varied with temperatures, viz., 62 and -20 mV at room temperature (MCD) compared to 116 and -4 mV at about 10 K (EPR). The midpoint potential of the iron-sulfur center obtained by low-temperature EPR was 286 mV. An abrupt conformational change occurred immediately after this center was fully reduced resulting in a change in EPR line shape. The potentials of the two half-reactions of ubiquinone were measured by following the semiquinone radical signal at 110 K and 23 degrees C. Potentials of 176 and 51 mV were found at low temperature, while values of 200 and 110 mV were observed at room temperature. The midpoint potential of cytochrome c1 was found to be pH independent. The potentials of cytochrome b were also independent of pH when titrations were performed in deoxycholate buffers, while a variation of -30 mV per pH unit was observed for both cytochrome c species in taurocholate buffers. These two detergents also produced different MCD contributions of the two b cytochromes. A decrease in Em of greater than 300 mV was found in potentiometric measurements of cytochrome c1 at high ratios of dye to Complex III. Antimycin does not affect the redox potentials of cytochrome c1 but appears to induce a transition of the low-potential b heme to a high-potential species. This transition is mediated by ubiquinone.     

Kinetic studies on the reaction of ethylenediaminetetraacetatochromium(III) complex with hydrogen peroxide

The rate of reaction of [Cr(III)Y]_aq (Y is EDTA anion) with hydrogen peroxide was studied in aqueous nitrate media [μ = 0.10 M (KNO₃)] at various temperatures. The general rate equation, Rate = $\text{k}{}_1\text{+}\text{k}{}_2\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}\text{1 +}\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}$ [Cr(III)Y]_aq[H₂O₂] holds over the pH range 5–9. The decomposition reaction of H₂O₂ is believed to proceed via two pathways where both the aquo and hydroxo-quinquedentate EDTA complexes are acting as the catalyst centres. Substitution-controlled mechanisms are suggested and the values of the second-order rate constants k₁ and k₂ were found to be 1.75 × 10^?2 M^?1 s^?1 and 0.174 M^?1 s^?1 at 303 K respectively, where k₂ is the rate constant for the aquo species and k₂ is that for the hydroxo complex. The respective activation enthalpies (ΔH^*₁ = 58.9 and ΔH^*₂ = 66.5 KJ mol^?1) and activation entropies (ΔS^*₁ = ?85 and ΔS^*₂ = ?40 J mol^?1 deg^?1) were calculated from a least-squares fit to the Eyring plot. The ionisation constant pK₁, was inferred from the kinetic data at 303 K to be 7.22. Beyond pH 9, the reaction is markedly retarded and ceases completely at pH ? 11. This inhibition was attributed in part to the continuous loss of the catalyst as a result of the simultaneous oxidation of Cr(III) to Cr(VI).     

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.