Escherichia coli flavohaemoglobin (Hmp) with equistoichiometric FAD and haem contents has a low affinity for dioxygen in the absence or presence of nitric oxide

A purification procedure for flavohaemoglobin Hmp (NO oxygenase) is described that gives high yields of protein with equistoichiometric haem and FAD contents. H2O2 accumulated on NADH oxidation by the purified protein and in cell extracts with elevated Hmp contents. H2O2 probably arose by dismutation from superoxide, which was also detectable during oxygen reduction: water was not a product. In the absence of agents that scavenge superoxide and peroxide, the mean K-m for oxygen was 80 muM; the addition of 15 muM FAD decreased the K-m for oxygen to 15 muM without a change in V-max but catalysed cyanide-insensitive oxygen consumption, attributed to electron transfer from favins to O-2. Purified Hmp consumed NO in the absence of added FAD (approx. 1 O-2 per NO), which is consistent with NO oxygenation. However: half-maximal rates of NO-stimulated O-2 consumption required approx. 47 muM O-2; NO removal was ineffective at physiologically relevant O-2 concentrations (below approx. 30 muM O-2). On exhaustion of O-2, NO was removed by a cyanide-sensitive process attributed to NO reduction, with a turnover number approx. 1%, of that for oxygenase activity. These results suggest that the ability of Hmp to detoxify NO might be compromised in hypoxic environments.