A study on nitrate reductase from Propionibacterium acidi-propionici.

Cell extract from a strain of Propionibacterium acidi-propionici with high nitrate reductase (NaR) activity catalyzed nitrate reduction with glycerol phosphate, NADH, or lactate. The reaction was inhibited partially by fumarate or oxygen. NaR linked to methyl viologen was found mostly in particulate fractions. It was solubilized by treatment with Emulgen 810 and purified 46-fold by DEAE-cellulose, Sepharose 4B, and triple DEAE-Sephadex chromatographies in the presence of the detergent. It was rather labile but was stabilized by glycerol. The molecular weight was estimated to be 230,000 by Sepharose 4B gel filtration and the isoelectric point was pH 5.0-5.5. The pH optimum was at 6.5-7.5 and Km for nitrate was 0.1 mM. As electron donors, methyl and benzyl viologen were utilized well but FAD and FMN were fairly ineffective. Chlorate was an active acceptor as well as nitrate. Azide, cyanide, and thiocyanate inhibited NaR. On adding 1 mM tungstate to the growing medium, the NaR level in grown cells was lowered; addition of 0.01 mM molybdate restored the activity partially. NaR is suggested to be a molybdo-protein, similar to this enzyme from other bacteria.