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) was 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 flavoprotein:ubiquinone oxidoreductase (ETF-UO) characterized in mammalian tissues. 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 titration on the enzyme with dithionite yielded an oxidation-reduction midpoint potential (Em) 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 Em values for the 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 direction opposite to that of the ETF-UO. Incubations of A. suum submitochondrial particles with NADH, 2-methylcrotonyl-CoA, purified A. suum electron-transfer flavoprotein and 2-methyl branched-chain enoyl-CoA reductase resulted in significant 2-methylbutyryl-CoA formation, which was inhibited by both rotenone and antisera to the purified ETF-RO. Quinone extraction of the submitchondrial particles with dry pentane resulted in almost the complete loss of 2-MBCoA formation by the system. However, the reincorporation of rhodoquinone, but not ubiquinone restored over 50% of the NADH-dependent 2-MBCoA formation.
Ma
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Department of Biology, University of Toledo, Ohio 43606.
Funk
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Dunham
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Komuniecki
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000693:Anaerobiosis; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D017165:Ascaris suum / Q000201:enzymology*; D002417:Cattle; D002845:Chromatography; D044927:Electron-Transferring Flavoproteins; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005420:Flavoproteins / Q000302:isolation & purification* / Q000378:metabolism*; D046911:Macromolecular Substances; D008297:Male; D008929:Mitochondria, Heart / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D008931:Mitochondria, Muscle / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism*; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism*; D011199:Potentiometry; D011817:Rabbits / Q000276:immunology; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013367:Submitochondrial Particles / Q000201:enzymology; D013552:Swine; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000378:metabolism
D000596:Amino Acids; D000906:Antibodies; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D014451:Ubiquinone; D010088:Oxidoreductases; C083272:rhodoquinone oxidoreductase; C003304:rhodoquinone