PURIFICATION AND CHARACTERIZATION OF PYRUVATE OXIDOREDUCTASE FROM THEPHOTOSYNTHETIC BACTERIUM RHODOBACTER-CAPSULATUS

Pyruvate:ferredoxin (flavodoxin) oxidoreductase (POR) was purified 305 0-fold to apparent homogeneity from the photosynthetic bacterium Rhodo bacter capsulatus using ion-exchange, Reactive Red, and gel filtration chromatography. The isolated enzyme was sensitive to dilution and oxy gen (especially when in dilute solution). The molecular mass of the na tive enzyme was determined by high performance liquid chromatography g el filtration to be 270 +/- 20 kDa. Since a subunit molecular mass of 130 +/- 5 kDa was found by denaturing gel electrophoresis, FOR from R capsulatus thus appears to be a homodimer. Electron paramagnetic reson ance analysis showed that a free radical was formed upon the addition of pyruvate. This FOR is shown to be an indiscriminate electron donor causing the full reduction of R capsulatus flavodoxin (Fld), R capsula tus ferredoxin I (FdI), R capsulatus ferredoxin II (FdII), as well as the major plant-type ferredoxin (FdI) from Anabaena variabilis. The pu rified 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 analy sis shows that R capsulatus FOR is constitutively synthesized, with sy nthesis augmented under nitrogen-fixing conditions (34 +/- 13%) and de creased in acetate and aerobically grown cells. (C) 1998 Published by Elsevier Science B.V. All rights reserved.