Purification and properties of a dissimilatory nitrite reductase of a denitrifying phototrophic bacterium

Abstract

Nitrite reductase [nitric-oxide : (acceptor) oxidoreductase, EC 1.7.2.1] from a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans, was purified. The molecular weight of the enzyme, estimated by gel-filtration, was 80,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified enzyme showed a single 39,000 molecular weight band, indicating that the enzyme was composed of two subunits of identical molecular weight. The oxidized form of the enzyme exhibited maximum absorption at 280 nm, 450 nm and 590 nm, and the reduced form only at 280 nm. The ESR spectrum of a frozen solution of the oxidized enzyme showed a typical spectrum pattern of a copper protein, suggesting that two types of Cu2+ existed within the enzyme. Estimates with an atomic absorption spectrophotometer, revealed two copper atoms per molecule. The optimum pH of the enzyme was 7.0. Km for nitrite was estimated to be 51 μM, and the optimum temperature, 30°C. The enzyme was inhibited by CO, potassium cyanide and diethyldithiocarbamate and activated by monoiodoacetate. Phenazine methosulfate, 2,6-dichlorophenolindophenol, horse heart cytochrome c, and cytochrome c2 from this bacterium were suitable electron donors. The enzyme also showed cytochrome c oxidase activity.