antimycin and duroquinol

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

A study is presented of the effect of Zn2+ on the enzymatic properties of the bovine-heart cytochrome-bc1 complex. Micromolar concentrations of Zn2+ reversibly inhibit the cytochrome-c reductase activity of either the cholate-solubilized or liposome-reconstituted complex. Kinetic analysis of the redox reactions of the cytochromes indicate that Zn2+ affects the activity of the complex at the quinol oxidation site. The following have been determined: (a) Zn2+ inhibits the pre-steady-state reduction of cytochrome c1 by duroquinol either in the absence or in the presence of antimycin, (b) it does not inhibit the reduction of b cytochromes in the absence of antimycin or in the presence of myxothiazol, (c) it inhibits cytochrome-b reduction in the presence of antimycin. Furthermore Zn2+ inhibits the antimycin-promoted oxidant-induced extrareduction of b cytochromes. Addition of Zn2+ to reduced bc1 complex causes a red shift in the absorption spectrum of cytochrome b566 and a substantial decrease in the signal intensity of the EPR spectrum of the Fe-S protein. This is interpreted as an interaction of Zn2+ with the 2Fe-2S-cluster region of the Fe-S protein, thus giving rise to inhibition of the reductase activity and of the antimycin-insensitive reduction route of b cytochromes. A Scatchard-plot of 65Zn2+ binding to the native isolated complex gave a straight line from which a value of three binding sites and a single dissociation constant of 3 x 10(-6) M can be calculated, which is practically equal to the concentration causing 50% inhibition of electron flow.

Topics: Animals; Antimycin A; Cations, Divalent; Cattle; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Electrophoresis, Polyacrylamide Gel; Ferricyanides; Hydroquinones; Kinetics; Mitochondria, Heart; NADH Dehydrogenase; Oxidation-Reduction; Protons; Spectrum Analysis; Zinc