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A method for measuring the kinetics of oxidation and reduction of endogenous Coenzyme Q in isolated Complex III from mitochondria has been developed for combining rapid-quenching and HPLC techniques. A time resolution of several milliseconds allows for the monitoring of redox events of both Q(,6) from yeast and Q(,10) from beef-heart during the initial phase of reduction of Complex III by Q(,1)H(,2), a soluble Coenzyme Q analog. Potent inhibition of the cytochrome c reductase activity of Complex III is obtained with antimycin, myxothiazol and 5-(n-undecyl)-6-hydroxyl-4,7-dioxobenzothiazole (UHDBT). These three inhibitors, and also funiculosin, were further studied by characterizing their effects on the visible absorbance, MCD, and epr spectra and the redox properties of the metal centers in the complex. Funiculosin alters the epr lineshape of the iron-sulfur cluster and that of cytochrome b; epr spectra recorded at 12(DEGREES) K reveal complete reduction of b(,562) by ascorbate. UHDBT also changes the lineshape of the iron-sulfur cluster and this change is partially reversed by myxothiazol. Funiculosin and UHDBT raise the midpoint potential of the iron-sulfur cluster by 157 and 74 mV, respectively. Only UHDBT changes the potential of c(,1), lowering it by 35 mV. The kinetics of reduction of cytochromes b and c(,1), monitored by absorbance increases at 561.5 and 553.5 nm, respectively, and Coenzyme Q(,6) of Complex III were examined. The absorbance at 561.5 nm increases most rapidly, followed by a slower decrease, correlating with the increase at 553.5 nm. Coenzyme Q(,6) was reduced very rapidly, reaching a near-equilibrium value of 30% within 8 milliseconds. Pre-reduction of the iron-sulfur cluster and c(,1) by ascorbate yields a biphasic, rapid absorbance increase at 561.5 nm, with no subsequent decrease. The presence of antimycin, funiculosin, myxothiazol or UHDBT also eliminates the secondary decrease at 561.5 nm, but only myxothiazol and UHDBT inhibit the increase in absorbance at 553.5 nm. Depletion of phospholipids and Coenzyme Q(,6) from Complex III drastically inhibits the absorbance increases at either wavelength. Linear, branched, and cyclic mechanisms of Complex III electron transfer were simulated by numerical-integration. The only model successful in predicting the pattern of reduction of Complex III by Q(,1)H(,2) is the Q cycle. Satisfactory simulations of reduction of oxidized, ascorbate-reduced, and myxothiazol- and UHDBT-inhibited Complex III were obtained. |
