Purification and characterization of pyruvate oxidoreductase from the photosynthetic bacterium Rhodobacter capsulatus.
Pyruvate:ferredoxin (flavodoxin) oxidoreductase (POR) was purified 3050-fold to apparent homogeneity from the photosynthetic bacterium Rhodobacter capsulatus using ion-exchange, Reactive Red, and gel filtration chromatography. The isolated enzyme was sensitive to dilution and oxygen (especially when in dilute solution). The molecular mass of the native enzyme was determined by high performance liquid chromatography gel filtration to be 270+/-20 kDa. Since a subunit molecular mass of 130+/-5 kDa was found by denaturing gel electrophoresis, POR from R. capsulatus thus appears to be a homodimer. Electron paramagnetic resonance analysis showed that a free radical was formed upon the addition of pyruvate. This POR is shown to be an indiscriminate electron donor causing the full reduction of R. capsulatus flavodoxin (Fld), R. capsulatus ferredoxin I (FdI), R. capsulatus ferredoxin II (FdII), as well as the major plant-type ferredoxin (FdI) from Anabaena variabilis. The purified enzyme can couple the oxidation of pyruvate to the reduction of nitrogenase in a coupled system with either R. capsulatus ferredoxins or nif-specific flavodoxin, NifF; (Fld>FdI>FdII). Immunoblot analysis shows that R. capsulatus POR is constitutively synthesized, with synthesis augmented under nitrogen-fixing conditions (34+/-13%) and decreased in acetate and aerobically grown cells.
Yakunin
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Département de Microbiologie et Immunologie, Université de Montréal, C.P. 6128, succursale Centre-ville, Montreal, Quebec H3C 3J7, Canada.
Hallenbeck
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D000114:Acetylene / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000502:physiology; D004795:Enzyme Stability; D005609:Free Radicals / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000737:chemistry*; D009591:Nitrogenase / Q000378:metabolism; D011487:Protein Conformation; D050743:Pyruvate Synthase; D019289:Pyruvic Acid / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D013053:Spectrophotometry
D000596:Amino Acids; D001426:Bacterial Proteins; D005609:Free Radicals; D019289:Pyruvic Acid; D009591:Nitrogenase; D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase; D000114:Acetylene