Direct spectrophotometric studies of the kinetics of oxidation and reduction of ubiquinone of heart-muscle particles

Abstract

An ultraviolet-extinction change is caused by addition of cyanide or antimycin A to a succinic-oxidase system. The magnitude of the change is proportional to the rate of production of fumarate. The spectrum representing the extinction change caused by addition of cyanide to the succinic-oxidase system has a peak near 275 mu and a shape resembling the difference spectrum of ethanolic solutions of reduced and oxidized ubiquinone derived from heart-muscle particles. The former spectrum is, however, shifted to longer wavelengths. The ultraviolet-extinction change is identified with the reduction of endogenous ubiquinone. No spectroscopic evidence for a semiquinone, free radical or otherwise altered reduction product has been obtained. The extinction change is incomplete in the presence of succinate alone, but goes rapidly to completion in the presence of cyanide or antimycin A. About 0.5-m[image]-succinate gives half-maximal reduction of ubiquinone in the cyanide-inhibited system. Shortly after the addition of succinate to the succinic-oxidase system, the reduction of ubiquinone is about 30% of the total reduction obtained in the presence of cyanide. This represents a steady-state reduction of ubiquinone that is less than that observed by the chemical extraction method and at longer times after adding succinate. No spectroscopically detectable ubiquinone is found on the oxygen side of the antimycin-sensitive point, and insufficient ubiquinone is available to act as an "intercytochrome carrier". Upon adding succinate to cyanide- or antimycin-treated particles, quinone is reduced in what appears to be a zero-order reaction for the first two-thirds of the reaction. The high activity of the heart-muscle preparations precluded accurate ubiquinone-reductase activity measurements by manual mixing at 26[degree] and studies in the temperature range 7-9-5[degree] are reported. The kinetics of reduction of endogenous ubiquinone (derived from the direct spectrophotometric data) agree to within 20% with those obtained by chemical assay on light-petroleum extracts of ubiquinone. The direct data are computed on the basis of A e 275-300 mu = 10 cm.-1 m[image] -1. The direct spectrophotometric and indirect chemical methods give ubiquinone -reductase rates at 7-9.5[degree] that are about one-third of the succinic-oxidase rates. A comparison of the reduction of ubiquinone and the production of fumarate made by the spectrophotometric method at 8[degree] shows that the maximum rate of ubiquinone reduction is one-third that of the steady-state rate of fumarate production. It follows that only if the value of ^ e 275-300 mu for ubiquinone were 3.3 cm.-1 m[image] -1 would the rates of ubiquinone reduction and fumarate production be equal. The effect of the "back" oxidation reaction upon ubiquinone-reductase activity is shown to be insignificant. On the basis of the chemical mechanism proposed here the kinetic discrepancies between ubiquinone-reductase activity and succinic-oxidase activity are irreconcilable, and so far no part of the total ubiquinone has been found to be reduced at a rate equal to that at which succinate is oxidized in the steady state.