Further Purification and Properties of Neurospora Nitrate Reductase

Abstract

Abstract Neurospora crassa (5297a) NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.2), a soluble, sulfhydryl-containing protein with FAD, a cytochrome b designated cytochrome b557 (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, FADH2-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 b557, was visible at the position corresponding to the major buffalo black-staining zone. By the use of sucrose density gradient centrifugation, a relative s20, w0.725 value of 8.0 for the nitrate reductase was found, and with Sephadex G-200 gel filtration techniques, a Stokes radius of 70 A was determined. From the relationship, mol wt = 6πeNas/(1 - vp), a molecular weight of 228,000 was calculated, assuming v = 0.725 cc per g. All enzymatic activities associated with nitrate reductase are heat-labile but to varying degrees, with the NADPH-nitrate and -cytochrome c reductases being most sensitive, and FADH2- and 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 b557 associated with nitrate reductase activity shows a typical cytochrome b type visible absorption spectrum at liquid nitrogen temperature and is a protoporphyrin IX heme as judged from the spectrum of its pyridine hemochromogen derivative. Studies with the inhibitor, p-hydroxymercuribenzoate, reveal the presence of one or more readily accessible sulfhydryl groups functioning between NADPH and FAD. FADH2-nitrate reductase and reduced methyl viologen-nitrate reductase are considerably less sensitive to this —SH reagent. Inhibition by metal-binding agents has suggested the possible involvement of a second metal moiety (in addition to the molybdenum), functioning earlier in the electron transfer scheme. No negative feedback phenomena at the enzymatic level could be found with the variety of nitrogen compounds tested.