PURIFICATION AND CHARACTERIZATION OF ELECTRON-TRANSFER FLAVOPROTEIN RHODOQUINONE OXIDOREDUCTASE FROM ANAEROBIC MITOCHONDRIA OF THE ADULT PARASITIC NEMATODE, ASCARIS-SUUM

Electron-transfer flavoprotein:rhodoquinone oxidoreductase (ETF-RO) wa s purified to homogeneity from anaerobic mitochondria of the parasitic nematode, Ascaris suum. The enzyme has a subunit molecular mass of 64 .5 kDa and is similar in many respects to the electron-transfer flavop rotein:ubiquinone oxidoreductase (ETF-UO) characterized in mammalian t issues. EPR spectroscopy of the purified enzyme revealed signals at g = 2.076, 1.936, and 1.883, arising from an iron-sulfur center, as well as signals attributable to a flavin semiquinone. Potentiometric titra tion on the enzyme with dithionite yielded an oxidation-reduction midp oint potential (E(m)) for the iron-sulfur center of +25 mV at pH 7.4. The reduction of flavin occurred in two distinct steps, with a flavin semiquinone radical detected as an intermediate. The E(m) values for t he two steps in the complete reduction of flavin were +15 mV and -9 mV , respectively. Physiologically, the ascarid ETF-RO accepts electrons from a low potential quinone, rhodoquinone, and functions in a directi on opposite to that of the ETF-UO. Incubations of A. suum submitochond rial particles with NADH, 2-methylcrotonyl-CoA, purified A. suum elect ron-transfer flavoprotein and 2-methyl branched-chain enoyl-CoA reduct ase resulted in significant 2-methylbutyryl-CoA formation, which was i nhibited by both rotenone and antisera to the purified ETF-RO. Quinone extraction of the submitochondrial particles with dry pentane resulte d in almost the complete loss of 2-MBCoA formation by the system. Howe ver, the reincorporation of rhodoquinone, but not ubiquinone, restored over 50% of the NADH-dependent 2-MBCoA formation.