hexacyanoferrate-iii and duroquinol

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

Membrane-permeable electron donors, duroquinol, diphenylcarbazide, pyrocatechol and tert-octylcatechol, promoted both reduction of an impermeant electron acceptor and proton transport with cultured carrot cells. These cells were preloaded with electron donors for 15, 30, 45 and 60 min. Aliquots of cells were removed at various times, washed free of excess electron donors and assayed for their effect on transplasma membrane redox with impermeable hexacyanoferrate (HCF III) as the electron acceptor and for simultaneous H+ excretion in the presence of hexacyanoferrate. All four electron donors stimulated HCF III reduction and associated H+ excretion. Below a rate of hexacyanoferrate reduction of 6 mumol/g dry wt. per min, the ratios of H+/e- were between 0.3 and 1 with low concentrations (0.1 mM) of the added electron donors. When hexacyanoferrate reduction exceeded 6 mumol/g dry wt. per min, proton release began to cascade to give ratios of 1 to 3, suggesting activation of an H(+)-ATPase or a proton transporter. This behavior by cultured carrot cells indicates that a certain threshold of proton concentration in a limited membrane domain must be reached in order for the proton channel to be opened.

Topics: Biological Transport, Active; Catechols; Cell Membrane; Cells, Cultured; Diphenylcarbazide; Electrons; Ferricyanides; Hydrogen; Hydroquinones; Ion Channels; Oxidation-Reduction; Plant Cells; Plants; Protons

The effect of the histidine-modifier ethoxyformic anhydride (EFA) on the enzymatic properties of the mitochondrial b-c1 complex (ubiquinol-cytochrome c reductase) has been investigated. Chemical modification by EFA inhibited to the same extent the reductase and the proton translocating activity of the complex. In particular EFA modification of the complex resulted in: strong inhibition of the antimycin-insensitive reduction of b cytochromes; inhibition of the antimycin-promoted oxidant-induced reduction of b cytochromes and inhibition of oxidation of pre-reduced b cytochromes. Analysis of the absorbance at 238 nm, indicative of N-(ethoxyformyl)histidine derivative, of the various polypeptide subunits separated by high-pressure liquid chromatography procedure, showed that EFA modified residues in core proteins and in the low-molecular-mass proteins. Both the inhibition of the redox and the protonmotive activity of the complex and the absorbance increase at 238 nm of the core protein fraction were readily reversed by hydroxylamine, indicating that modification of histidine residue(s) in core protein(s) is critical for the activity of the complex. This was supported by the finding that modification of the reductase with EFA prevented binding of fluorescein isothiocyanate to histidine residue(s) in core protein II. EFA modification of the reductase was without effect on the binding of N-(7-dimethylamino-4-methylcoumarinyl)maleimide to the various polypeptides of the complex except for the binding to the Fe-S protein which was greatly potentiated. Thus primary chemical modification of histidine residue(s) in core protein (II) appears to cause, in turn, a conformational change in the Rieske Fe-S protein.

Topics: Animals; Cattle; Chromatography, High Pressure Liquid; Diethyl Pyrocarbonate; Electron Transport; Electron Transport Complex III; Ferricyanides; Formates; Hydroquinones; Kinetics; Mitochondria, Heart; Oxidation-Reduction

Topics: Animals; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Ethylmaleimide; Ferricyanides; Hydrogen; Hydroquinones; Mitochondria, Liver; NAD; Oxygen Consumption; Potassium; Potassium Cyanide; Rats; Rotenone; Succinates; Succinic Acid; Valinomycin