Respiratory nitrate reductase from Paracoccus denitrificans

Abstract

The b-type haem centers of the three (.alpha., .beta. and .gamma.) subunit nitrate reductase from Paracoccus denitrificans have been analyzed by redox potentiometry. Two components were identified with mid-point potentials +95 mV and +210 mV. Washing, in the absence of Mg2+ ions, of cytoplasmic membrane vesicles from P. denitrificans promoted selective release of nitrate reductase activity. The released enzyme was purified by chromatography and shown to contain .alpha. and .beta., but not .gamma. polypeptides. A haem spectrum was absent, consistent with the lack of the .gamma. subunit. The .alpha. and .beta. polypeptides of the water-soluble nitrate reductase had molecular masses that were identical to those of the detergent-purified enzyme and also of the nitrate reductase in cytoplasmic membranes. The observation, together with the failure of protease inhibitors to prevent release from the membrane, indicates that the release is not related to limited proteolysis of the .alpha. and/or .beta. polypeptides. The relative molecular mass of the water-soluble .alpha..beta. enzyme was estimated to be approximately 200,000. The water-soluble nitrate reductase was released from intact inverted cytoplasmic membrane vesicles as jugded by loss of NADH-NO3- reductase activity and retention by the vesicles after washing of uncoupler-sensitive NADH-oxidase activity. These observations show that .alpha. and .beta. polypeptides, and therefore the active site for nitrate reduction, are located on the cytoplasmic side of the membrane. Attempts to reverse the nitrate reductase activity of the enzyme, using nitrite as reductant plus ferricyanide or chlorate as tested oxidants, were unsuccessful. The implications for the mechanism of the enzyme are discussed.