Purification and properties of succinate-ubiquinone oxidoreductase complex from Paracoccus denitrificans.
Highly active succinate-ubiquinone reductase has been purified from cytoplasmic membranes of aerobically grown Paracoccus denitrificans. The purified enzyme has a specific activity of 100 units per mg protein, and a turnover number of 305 s-1. Succinate-ubiquinone reductase activity of the purified enzyme is inhibited by 3'-methylcarboxin and thenoyltrifluoroacetone. Four subunits, with apparent molecular masses of 64.9, 28.9, 13.4 and 12.5 kDa, were observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains 5.62 nmol covalently bound flavin and 3.79 nmol cytochrome b per mg protein. The 64.9 kDa subunit was shown to be a flavoprotein by its fluorescence. Polyclonal antibodies raised against this protein cross-reacted with the flavoprotein subunit of bovine heart mitochondrial succinate-ubiquinone reductase. The 28.9 kDa subunit is likely analogous to the bovine heart iron protein, and the cytochrome b heme is probably associated with one or both of the low-molecular-weight polypeptides. The cytochrome b is not reducible with succinate but is reoxidized with fumarate after prereduction with dithionite. Iron-sulfur clusters S-1 and S-3 of the Paracoccus oxidoreductase exhibit EPR spectra very similar to their mitochondrial counterparts. Paracoccus succinate-ubiquinone reductase complex is thus similar to the bovine heart mitochondrial enzyme with respect to prosthetic groups, enzymatic activity, inhibitor sensitivities, and polypeptide subunit composition.
Pennoyer
J D
JD
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03756.
Ohnishi
T
T
Trumpower
B L
BL
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D003573:Cytochrome b Group / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D042963:Electron Transport Complex II; D004591:Electrophoresis, Polyacrylamide Gel; D005415:Flavins / Q000032:analysis; D007118:Immunoassay; D046911:Macromolecular Substances; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010231:Paracoccus denitrificans / Q000201:enzymology*; D010455:Peptides / Q000032:analysis; D013053:Spectrophotometry; D013385:Succinate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000494:pharmacology
D003573:Cytochrome b Group; D005415:Flavins; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D010455:Peptides; D014451:Ubiquinone; D010088:Oxidoreductases; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase