ISOLATION AND CHARACTERIZATION OF THE PYRUVATE-FERREDOXIN OXIDOREDUCTASE FROM THE SULFATE-REDUCING BACTERIUM DESULFOVIBRIO-AFRICANUS

We report the first purification and characterization of a pyruvate-fe rredoxin oxidoreductase (FOR) from a sulfate-reducing bacterium, Desul fovibrio africanus. The enzyme as isolated is highly stable in the pre sence of oxygen and exhibits a specific activity of 14 U/mg. D. africa nus POR is a 256 kDa homodimer which contains thiamine pyrophosphate ( TPP) and iron-sulfur clusters. EPR spectroscopic study of the enzyme i ndicates the presence of three [4Fe-4S](2+/1+) centers/subunit. The mi dpoint potentials of the three centers are -390 mV, -515 mV and -540 m V. The catalytic mechanism of FOR involves a free radical intermediate which disappears when coenzyme A is added. This behaviour is discusse d in terms of an electron-transport chain from TPF to the acceptor. Th e enzyme activated by dithioerythritol shows an exceptionally high act ivity compared with other mesophile PORs and becomes very sensitive to oxygen in contrast to the enzyme before activation. The comparison of EPR spectra given by the as isolated and activated enzymes shows that neither the nature, nor the arrangement of FeS centers are affected b y the activation process. D. africanus ferredoxins I and II are involv ed as the physiological electron carriers of the enzyme. FOR was shown to be located in the cytoplasm by immunogold labelling.