Purification and characterization of the assimilatory nitrate reductase of Azotobacter vinelandii

Abstract

1. A soluble reduced Methyl Viologen-dependent assimilatory nitrate reductase from Azotobacter vinelandii strain UW136 grown aerobically on nitrate was purified to homogeneity by the criteria of nitrate reductase activity staining, and coincidence of a Coomassie Blue-staining protein band on polyacrylamide gels run under non-denaturing conditions. The specific activity was 3 μmol of NO2- formed/min per mg of protein. 2. Gel filtration on Superose-12 and SDS/PAGE showed that the enzyme had an M(r) of 105000 and was monomeric. The enzyme contained 1 Mo atom, 4 Fe atoms and 4 acid-labile sulphide atoms per molecule; no evidence for the presence of cytochrome or FAD was found. 3. Mo was present in a molybdenum cofactor, which on extraction was capable of activating apo-(nit-1) nitrate reductase present in crude extracts of nit-1 mutants of Neurospora crassa. 4. As isolated, the enzyme had e.p.r. signals assigned to Mo(V) with g-values g1 = 2.023; g2 = 1.998; g3 = 1.993 and with g(av) = 2.004 indicating an unusual environment of Mo in this enzyme. 5. Reduction with S2O42- bleached the e.p.r. signals which, on reoxidation after the addition of NO33- to initiate enzyme turnover, exhibited at short times Mo(V) signals similar to those of dissimilatory nitrate reductases, with g1 = 1.998; g2 = 1.989; g3 = 1.981 and g(av) = 1.989. Prolonged incubation subsequently gave a mixture of both e.p.r. species. 6. Neither NADH nor NADPH was effective as an electron donor, but reduced Methyl Viologen (apparent K(m) 988 μM) and reduced Bromophenol Blue (apparent K(m) 158 μM) were effective. With these donors the apparent Km values for nitrate were 70 μM and 217 μM respectively.