Electron transfer flavoprotein-ubiquinone oxidoreductase from pig liver: purification and molecular, redox, and catalytic properties

Abstract

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) was purified to homogeneity from pig liver submitochondrial particles. It is comparable in MW and general properties to ETF-QO from beef heart [Ruzicka, F.J., and Beinert, H. (1977) J. Biol. Chem. 252, 8440-8445], and the ESR signals of the reduced ion-sulfur cluster are essentially identical. ETF-QO catalyzes the transfer of electrons from electro-transfer flavoprotein (ETF) to nitro blue tetrazolium, with a sluggish reaction turnover number of .apprx. 10-30 min-1. In contrast, the enzyme rapidly disproportionates ETF semiquinone, with a turnover number of 200 s-1. The reverse reaction, comproportionation of oxidized and hydroquinone ETF, provides an enzymatic assay for ETF-QO with picomolar sensitivity. Equilibrium spectrophotometric titrations show that ETF-QO accepts a maximum of 2 electrons from ETF and accepts 3 electron equivalents from dithionite or by photochemical reduction. All electrons from the enzymatically or chemically reduced protein can be transferred to 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone (PB), and this reaction is readily reversible. Reduction of ETF-QO by 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzohydroquinone is pH dependent and indicates the enzyme to have a redox potential that decreases by 47 mV per pH unit. Therefore, ETF-QO binds 1 to 2 protons upon reduction. E0'' at pH 7.3 is 38 mV. The ability of ETF-QO to catalyze the equilibration of ETF redox states has been used to evaluate the equilibrium 2ETFsq + nH+ .dblarw. ETFox + ETFhq. The pH dependence of the equilibrium indicates that n = 1 and is consistent with the assignment of ETF semiquinone (ETFsq) and hydroquinone flavin species as true anions. The 1-electron reduction potential of oxidized ETF is predicted to be independent of pH.