H218O isotope exchange studies on the mechanism of reduction of nitric oxide and nitrite to nitrous oxide by denitrifying bacteria. Evidence for an electrophilic nitrosyl during reduction of nitric oxide.

Reduction of NO and NO2-by whole cells of eight strains of denitrifying bacteria known to contain either heme cd1 or copper-containing nitrite reductases (NiRs) has been examined in the presence of H218O. All organisms containing heme cd1 NiRs exhibited relatively large extents of exchange between NO2- and H218O (39-100%), as monitored by the 18O content of product N2O. Organisms containing copper NiRs gave highly variable results, with Achromobacter cycloclastes and Pseudomonas aureofaciens exhibiting no 18O incorporation and Rhodopseudomonas sphaeroides and Alcaligenes entrophus exhibiting complete exchange between NO2- and H218O. Organisms containing heme cd1 NiRs exhibited significant but lower levels of exchange between NO and H218O than between NO2- and H218O, while organisms containing copper NiRs gave significantly higher amounts of 18O incorporation than observed for the heme cd1 organisms. These results demonstrate the existence of an NO-derived species capable of undergoing O-atom exchange with H218O during the reduction of NO. Trapping experiments with 15NO, 14N3-, and crude extracts of R. sphaeroides support the electrophilic nature of this intermediate and suggest its formulation as an enzyme nitrosyl, E-NO+, analogous to that observed during reduction of NO2-. The observation of lower levels of 18O incorporation with NO2- than with NO as substrate for A. cycloclastes and P. aureofaciens indicates that, for these organisms at least, a sequential pathway involving free NO as an intermediate is significantly less important than a direct pathway in which N2O is formed via reaction of two NO2- ions on a single enzyme.