Further purification and properties of Neurospora nitrate reductase.

Neurospora ctassa (5297a) NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.2), a soluble, sulfhydryl-containing protein with FAD, a cytochrome b designated cytochrome bsm (N. crassa), and molybdenum as prosthetic groups, has been purified 500-fold by procedures including pH adjustment, ammonium sulfate fractionation of the resultant supernatant solution, phase separation to remove nucleic acids, diethylaminoethyl cellulose chromatography, hydroxylapatite chromatography, and, finally, Sephadex G-200 gel filtration. Other enzymatic activities associated with NADPH-nitrate reductase throughout the purification and maintaining a proportional relationship with it are NADPH-cytochrome c reductase, FADHz-nitrate reductase, and reduced methyl viologen-nitrate reductase. Polyacrylamide gel electrophoresis of the most highly purified enzyme preparations yielded a major buffalo black-staining zone containing the above enzymatic activities, a somewhat smaller zone, and several faint zones. In unstained gels, a red zone, which was apparently due to the presence of cytochrome bcG7, was visible at the position corresponding to the major buffalo black-staining zone. By the use of sucrose density gradient centrifugation, a relative st,‘: value of 8.0 for the nitrate reductase was found, and with Sephadex G-ZOO gel f&ration techniques, a Stokes radius of 70 A was determined. From the relationship, mol wt = 6nqNas/(l cp), a molecular weight of 228,000 was calculated, assuming p = 0.725 cc per g. All enzymatic activities associated with nitrate reductase are heat-labile but to varying degrees, with the NADPHnitrate and -cytochrome c reductases being most sensitive, and FADH2and reduced methyl viologen-nitrate reductase activities being progressively less SO, in that order. The reduced methyl viologen-nitrate reductase activity showed a marked increase in activity as the NADPH activities declined. The cytochrome b5sT associated with nitrate reductase activity shows a typical cytochrome b type visible absorption spectrum at liquid nitrogen temperature and is a proto-