Electron-transfer flavoprotein-ubiquinone oxidoreductase from pig liver: purification and molecular, redox, and catalytic properties.
Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) was purified to homogeneity from pig liver submitochondrial particles. It is comparable in molecular weight and general properties to ETF-QO from beef heart [Ruzicka, F. J., & Beinert, H. (1977) J. Biol. Chem. 252, 8440-8445], and the electron spin resonance signals of the reduced iron-sulfur cluster are essentially identical. ETF-QO catalyzes the transfer of electrons from electron-transfer flavoprotein (ETF) to nitro blue tetrazolium, with a sluggish reaction turnover number of about 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 two electrons from ETF and accepts three 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 one to two protons upon reduction. The EO' 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+ in equilibrium ETFox + ETFhq.(ABSTRACT TRUNCATED AT 250 WORDS)
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004579:Electron Transport; D044927:Electron-Transferring Flavoproteins; D044943:Fatty Acid Desaturases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D008433:Mathematics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010743:Phospholipids / Q000032:analysis; D013053:Spectrophotometry; D013367:Submitochondrial Particles / Q000201:enzymology*; D013552:Swine
D000596:Amino Acids; D044927:Electron-Transferring Flavoproteins; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D010743:Phospholipids; D007501:Iron; D044943:Fatty Acid Desaturases; D000587:Oxidoreductases Acting on CH-NH Group Donors; C023120:electron-transferring-flavoprotein dehydrogenase