Abstract
Nitrate reductase (NaR) linked to reduced methyl viologen from Clostridium perfringens was purified by ammonium sulfate precipitation, DEAE-cellulose chromatography, disc electrophoresis on polyacrylamide gel, and triple DEAE-Sephadex chromatography. The specific activity was increased 1, 200-fold with a yield of 9%. The purified preparation was nearly homogeneous in disc electrophoresis. It was brown, and its spectrum showed a slight shoulder near 420nm as well as a peak at 280nm. The molecular weight was found to be 90, 000 based on s02, w (5.8 S) and 80, 000 by Sephadex G-100 gel filtration. In SDS-polyacrylamide electrophoresis, it showed only a single band with a molecular weight of 90, 000; it had no subunit structure. The isoelectric point was pH 5.5, and the optimum pH was 9. Mn2+, Fe22+, Mg2+, and Ca2+ stimulated the activity. Km for nitrate was 0.10mM, and nitrate was stoichiometrically reduced to nitrite in the presence of 2mM Mn2+. Ferredoxin fraction obtained from extracts of the bacterium was utilizable as an electron donor at pH 8. Cyanide and azide inhibited the enzyme. The formation of NaR was induced by nitrate and inhibited by 0.5mM tungstate, but recovered in the presence of 0.1mM molybdate; NaR of C. perfringens appears to be a molybdo-iron-sulfur protein.
