O2 regulation of denitrification in Flexibacter canadensis

We studied the sensitivity to oxygen of the reductases involved in denitrification by whole cells and membrane fractions of Flexibacter canadensis. All of the nitrate reductase activity was found in the membrane fraction, suggesting that the nitrate reductase of F. canadensis is largely or entirely a membrane-bound enzyme. Methyl viologen and benzyl viologen were good electron donors to nitrate reductase in both whole cells and membrane fractions, whereas glucose and glycerol were effective in whole cells but, as expected, not in membrane fractions. Oxygen, generated by means of H2O2 plus catalase, inhibited the production of nitrite from nitrate by intact cells but not by membrane fractions, suggesting that O2 exerts its inhibitory effect at the level of nitrate transport rather than nitrate reduction. In intact cells, the rates of nitric oxide accumulation during reduction of nitrite in the presence of 20 μM carbonyl cyanide m-chlorophenylhydrazone, and consumption of nitric oxide and nitrous oxide, decreased as the concentration of H2O2 was increased. The concentrations of H2O2 giving 50% inhibition of reduction of nitrate and nitrite were 0.34 and 0.12 mM, respectively. In contrast, the rates of nitric oxide and nitrous oxide consumption were inhibited by only 36 and 32% at a concentration of H2O2 of 3.99 mM. These results indicate that the reduction of both nitric oxide and nitrous oxide is relatively tolerant to oxygen, and that nitrite reductase is much more sensitive to oxygen than the other reductases.Key words: nitrate reductase, nitrate transport, denitrification, O2 inhibition, Flexibacter canadensis.