ubiquinone and duroquinol

In this focused review, we portray the recently reported 2.5 Å cyro-EM structure of the particulate methane monooxygenase (pMMO) from M. capsulatus (Bath). The structure of the functional holo-pMMO near atomic resolution has uncovered the sites of the copper cofactors including the location of the active site in the enzyme. The three coppers seen in the original X-ray crystal structures of the enzyme are now augmented by additional coppers in the transmembrane domain as well as in the water-exposed C-terminal subdomain of the PmoB subunit. The cryo-EM structure offers the first glimpse of the catalytic machinery capable of methane oxidation with high selectivity and efficiency. The findings are entirely consistent with the biochemical and biophysical findings previously reported in the literature, including the chemistry of hydrocarbon hydroxylation, regeneration of the catalyst for multiple turnovers, and the mechanism of aborting non-productive cycles to ensure kinetic competence.

Topics: Biocatalysis; Catalytic Domain; Copper; Hydroquinones; Methane; Methylococcus capsulatus; NAD; Oxidation-Reduction; Oxygenases; Protein Conformation, alpha-Helical; Protein Domains; Protein Subunits; Ubiquinone

The long-lived mutant of Caenorhabditis elegans, clk-1, is unable to synthesize ubiquinone, CoQ(9). Instead, the mutant accumulates demethoxyubiquinone(9) and small amounts of rhodoquinone(9) as well as dietary CoQ(8). We found a profound defect in oxidative phosphorylation, a test of integrated mitochondrial function, in clk-1 mitochondria fueled by NADH-linked electron donors, i.e. complex I-dependent substrates. Electron transfer from complex I to complex III, which requires quinones, is severely depressed, whereas the individual complexes are fully active. In contrast, oxidative phosphorylation initiated through complex II, which also requires quinones, is completely normal. Here we show that complexes I and II differ in their ability to use the quinone pool in clk-1. This is the first direct demonstration of a differential interaction of complex I and complex II with the endogenous quinone pool. This study uses the combined power of molecular genetics and biochemistry to highlight the role of quinones in mitochondrial function and aging.

Topics: Animals; Ascorbic Acid; Caenorhabditis elegans; Electron Transport Complex I; Electron Transport Complex II; Glutamic Acid; Hydroquinones; Malates; Mitochondria; Mutation; Oxidative Phosphorylation; Pyruvic Acid; Quinones; Substrate Specificity; Tetramethylphenylenediamine; Time Factors; Ubiquinone

Complex III in the mitochondrial electron transport chain is a proposed site for the enhanced production of reactive oxygen species that contribute to aging in the heart. We describe a defect in the ubiquinol binding site (Q(O)) within cytochrome b in complex III only in the interfibrillar population of cardiac mitochondria during aging. The defect is manifested as a leak of electrons through myxothiazol blockade to reduce cytochrome b and is observed whether cytochrome b in complex III is reduced from the forward or the reverse direction. The aging defect increases the production of reactive oxygen species from the Q(O) site of complex III in interfibrillar mitochondria. A greater leak of electrons from complex III during the oxidation of ubiquinol is a likely mechanism for the enhanced oxidant production from mitochondria that contributes to aging in the rat heart.

Topics: Aging; Animals; Antimycin A; Binding Sites; Cytochrome b Group; Electron Transport; Electron Transport Complex III; Enzyme Activation; Hydroquinones; In Vitro Techniques; Male; Methacrylates; Mitochondria, Heart; Mitochondrial Diseases; Myofibrils; Oxidation-Reduction; Polyenes; Rats; Reactive Oxygen Species; Thiazoles; Ubiquinone

The steady-state reduction of exogenous ubiquinone-2 by duroquinol as catalysed by the ubiquinol: cytochrome c oxidoreductase was studied in bovine heart mitoplasts. The reduction of ubiquinone-2 by duroquinol proceeds both in the absence of inhibitors of the enzyme, in the presence of outside inhibitors, e.g., myxothiazol, and in the presence of inside inhibitors, e.g., antimycin, but not in the presence of both inside and outside inhibitors. It is concluded that both the Qin-binding domain and the Qout-binding domain may independently catalyse this reaction. The rate of the reduction of ubiquinone-2 by duroquinol via the Qin-binding domain is dependent on the type of outside inhibitor used. The maximal rate obtained for the reduction of ubiquinone-2 by DQH2 via the Qout-binding domain, measured in the presence of antimycin, is similar to that catalysed by the Qin-binding domain of the non-inhibited enzyme and depends on the redox state of the high-potential electron carriers of the respiratory chain. The reduction of ubiquinone-2 by DQH2 via the Qin-binding domain can be described by a mechanism in which duroquinol reduces the enzyme, upon which the reduced enzyme is rapidly oxidized by ubiquinone-2 yielding ubiquinol-2. By determination of the initial rate under various conditions and simulation of the time course of reduction of ubiquinone-2 using the integrated form of the steady-state rate equation the values of the various kinetic constants were calculated. During the course of reduction of ubiquinone-2 by duroquinol in the presence of outside inhibitors only cytochrome b-562 becomes reduced. At all stages during the reaction, cytochrome b-562 is in equilibrium with the redox potential of the ubiquinone-2/ubiquinol-2 couple but not with that of the duroquinone/duroquinol couple. At low pH values, cytochrome b-562 is reduced in a single phase; at high pH separate reduction phases are observed. In the absence of inhibitors three reduction phases of cytochrome b-562 are discernible at low pH values and two at high pH values. In the presence of antimyin cytochrome b becomes reduced in two phases. Cytochrome b-562 is reduced in the first phase and cytochrome b-566 in the second phase after substantial reduction of ubiquinone-2 to ubiquinol-2 has occurred. In ubiquinone-10 depleted preparations, titration of cytochrome b-562, in the presence of myxothiazol, with the duroquinone/duroquinol redox couple yields a value of napp = 2, both at low and high pH.(

Topics: Animals; Antimycin A; Benzoquinones; Binding Sites; Cattle; Cytochrome b Group; Electron Transport; Electron Transport Complex III; Hydrogen-Ion Concentration; Hydroquinones; Methacrylates; Myocardium; Oxidation-Reduction; Thiazoles; Ubiquinone

Dimeric ubiquinol:cytochrome c reductase of Neurospora mitochondria was isolated as a protein-Triton complex and free of ubiquinol (Q). The enzyme was incorporated into phosphatidylcholine membranes together with Q. The effects of varying the molar ratio of Q to enzyme on the electron transfer from duroquinol (DHQ2) to the cytochromes c, c1 and b were studied. The rate of electron flow from DQH2 to cytochrome c was 15 times increased by Q and was maximal when one molecule of Q was bound to one enzyme dimer. The apparent Km value for DQH2 of the Q-free enzyme was 5 microM and of the Q-supplemented enzyme 25 microM. The pre-steady-state rate of electron transfer from DQH2 to cytochrome c1 was also 15 times increased by Q and was maximal with one Q molecule bound to one enzyme dimer. This effect of Q was inhibited by antimycin. The pre-steady-state rate of electron transfer from DQH2 to cytochrome b was 5 times decreased when Q was bound to the enzyme and this effect of Q was insensitive to myxothiazol. The H+/2e- stoichiometry with DQH2 as substrate of the Q-supplemented enzyme was 3.6. These results are interpreted in accordance with a Q-cycle mechanism operating in a dimeric cytochrome reductase. Each enzyme monomer catalyses a single electron transfer from the QH2-oxidation centre to the Q-reduction centre and the two monomers cooperate in the reduction of Q to QH2 at one Q-reduction centre. This centre contains two different binding sites for Q. DQH2 does not properly react at the QH2-oxidation centre. DQH2, however, binds to the loose Q-binding site of the Q-reduction centre and reduces the Q bound to the tight Q-binding site of the centre. The QH2 thus formed at the Q-reduction centre serves as electron donor for the QH2-oxidation centre.

Topics: Biological Transport; Cytochrome c Group; Electron Transport; Electron Transport Complex III; Hydroquinones; Mitochondria; Multienzyme Complexes; Neurospora; Oxidation-Reduction; Protons; Quinone Reductases; Ubiquinone

The respiratory nitrate reductase from Paracoccus denitrificans has been purified in the non-ionic detergent Nonidet P-40. The enzyme comprises three polypeptides, alpha, beta and gamma with estimated relative molecular masses of 127 000, 61 000 and 21 000. Duroquinol or reduced-viologen compounds acted as the reducing substrates. The nitrate reductase contained a b-type cytochrome that was reduced by duroquinol and oxidised by nitrate. A preparation of the enzyme that lacked both detectable b-type cytochrome and the gamma subunit was obtained from a trailing peak of nitrate reductase activity collected from a gel filtration column. Absence of the gamma subunit correlated with failure to use duroquinol as reductant; activity with reduced viologens was retained. It is concluded that in the plasma membrane of P. denitrificans the gamma subunit catalyses electron transfer to the alpha and beta subunits of nitrate reductase from ubiquinol which acts as a branch point in the respiratory chain. A new assay was introduced for both nitrate and quinol-nitrate oxidoreductase activity. Diaphorase was used to couple the oxidation of NADH to the production of duroquinol which acted as electron donor to nitrate reductase. Under anaerobic conditions absorbance changes at 340 nm were sensitive to nitrate concentrations in the low micromolar range. This coupled assay was used to determine that the purified enzyme had Km(NO-3) of 13 microM and a Km of 470 microM for ClO-3, an alternative substrate. With viologen substrates Km(NO-3) of 283 microM and Km(ClO-3) of 470 microM were determined; the enzymes possessed a considerably higher Vmax with either NO-3 or ClO-3 than was found when duroquinol was substrate. Azide was a competitive inhibitor of nitrate reduction in either assay system (Ki = 0.55 microM) but 2-n-heptyl-4-hydroxyquinoline N-oxide was effective only with the complete three-subunit enzyme and duroquinol as substrate, consistent with a site of action for this inhibitor on the b-type cytochrome. The low Km for nitrate observed in the duriquinol assay is comparable with the apparent Km(NO-3) recently reported for intact cells of P. denitrificans [Parsonage, D., Greenfield, A. J. & Ferguson, S. J. (1985) Biochim. Biophys. Acta 807, 81-95]. This similarity is discussed in terms of a possible requirement for a nitrate transport system. The nitrate reductase system from P. denitrificans is compared with that from Escherichia coli.

Topics: Chromatography, Gel; Counterimmunoelectrophoresis; Electrophoresis, Polyacrylamide Gel; Hydroquinones; Kinetics; Molecular Weight; Nitrate Reductase; Nitrate Reductases; Nitrates; Oxygen Consumption; Paracoccus denitrificans; Spectrum Analysis; Ubiquinone