Production and consumption of nitric oxide by denitrifyingFlexibacter canadensis

Abstract

Production and consumption of nitric oxide (NO) by Flexibacter canadensis cells under anaerobic conditions was investigated using a chemiluminescence NO analyzer. Net NO production from nitrite in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) was pH dependent, increased in the pH range from 4.5 to 6.5, and sharply decreased at pH >6.5. CCCP inhibited NO consumption but only at pH values ≤6.5. This can explain why CCCP stimulation of NO production depends on the pH. Denitrification of nitrite at high concentrations (≥5 mM) also resulted in net NO accumulation. Diethyldithiocarbamate, a copper chelating agent, prevented not only net production of NO during the reduction of nitrite in the presence of CCCP, but also production of nitrous oxide (N2O) from nitrite in the presence of C2H2. This suggests that F. canadensis may possess a copper-type nitrite reductase. However, cytochrome cd1- and copper-containing nitrite reductase DNA probes from Pseudomonas species did not hybridize with the total DNA of F. canadensis, indicating that the nitrite reductase of F. canadensis may possess unique properties. In addition to diethyldithiocarbamate, sulfide, carbon monoxide, azide, cyanide, hydroxylamine and Triton X-100 prevented net NO production from nitrite in the presence of CCCP, and also inhibited NO consumption. C2H2, an inhibitor of N2O reductase, did not affect NO production or consumption.Key words: nitrite reductase, nitric oxide (NO), carbonyl cyanide m-chlorophenylhydrazone (CCCP), Flexibacter canadensis.