The denitrifying nitrite reductase of Bacillus halodenitrificans

Abstract

Denitrifying nitrite reductase was purified from Bacillus halodenitrificans, a newly isolated moderate halophile, and identified as a dimeric nonheme copper protein with a molecular mass of 82 kDa comprising two identical 40-kDa subunits. Unlike its counterparts in other denitrifiers, this enzyme is firmly bound in the cytoplasmic membrane, a characteristic that may be related to the Gram-positive nature of this bacillus (and thus lack of a periplasmic space). The enzyme functioned most effectively at pH 6, and was activated by elevated salt concentrations, a property shared by the copper nitrite reductase from another, but nonhalophilic, denitrifier, Achromobacter cycloclastes. In contrast, high salt activation was not exhibited by cytochrome cd1 nitrite reductases from either nonhalophilic Thiobacillus denitrificans or halophilic Paracoccus halodenitrificans. The enzyme was rich in aspartate/asparagine and methionine residues. Electron paramagnetic resonance spectroscopy revealed both type 1 and type 2 copper in the protein. The physiological electron donor that transfers electrons to the copper nitrite reductase from A. cycloclastes, a small blue copper protein, did not transfer electrons to the copper nitrite reductase of B. halodenitrificans.