Partial purification and characterization of glutaryl-coenzyme A dehydrogenase, electron transfer flavoprotein, and electron transfer flavoprotein-Q oxidoreductase from Paracoccus denitrificans.
Glutaryl-coenzyme A (CoA) dehydrogenase and the electron transfer flavoprotein (ETF) of Paracoccus denitrificans were purified to homogeneity from cells grown with glutaric acid as the carbon source. Glutaryl-CoA dehydrogenase had a molecular weight of 180,000 and was made up of four identical subunits with molecular weights of about 43,000 each of which contained one flavin adenine dinucleotide molecule. The enzyme catalyzed an oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA, was maximally stable at pH 5.0, and lost activity readily at pH values above 7.0. The enzyme had a pH optimum in the range of 8.0 to 8.5, a catalytic center activity of about 960 min-1, and apparent Michaelis constants for glutaryl-CoA and pig liver ETF of about 1.2 and 2.5 microM, respectively. P. denitrificans ETF had a visible spectrum identical to that of pig liver ETF and was made up of two subunits, only one of which contained a flavin adenine dinucleotide molecule. The isoelectric point of P. denitrificans ETF was 4.45 compared with 6.8 for pig liver ETF. P. denitrificans ETF accepted electrons not only from P. denitrificans glutaryl-CoA dehydrogenase, but also from the pig liver butyryl-CoA and octanoyl-CoA dehydrogenases. The apparent Vmax was of similar magnitude with either pig liver or P. denitrificans ETF as an electron acceptor for these dehydrogenases. P. denitrificans glutaryl-CoA dehydrogenase and ETF were used to assay for the reduction of ubiquinone 1 by ETF-Q oxidoreductase in cholate extracts of P. denitrificans membranes. The ETF-Q oxidoreductase from P. denitrificans could accept electrons from either the bacterial or the pig liver ETF. In either case, the apparent Km for ETF was infinitely high. P. denitrificans ETF-Q oxidoreductase was purified from contaminating paramagnets, and the resultant preparation had electron paramagnetic resonance signals at 2.081, 1.938, and 1.879 G, similar to those of the mitochondrial enzyme.
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins / Q000302:isolation & purification / Q000378:metabolism*; D050770:Glutaryl-CoA Dehydrogenase; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008970:Molecular Weight; D009097:Multienzyme Complexes; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010231:Paracoccus denitrificans / Q000201:enzymology*; D013552:Swine
D000596:Amino Acids; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050770:Glutaryl-CoA Dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; C023120:electron-transferring-flavoprotein dehydrogenase