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 Km for oxygen was 80µM; the addition of 15µM FAD decreased the Km for oxygen to 15µM without a change in Vmax but catalysed cyanide-insensitive oxygen consumption, attributed to electron transfer from flavins to O2. Purified Hmp consumed NO in the absence of added FAD (approx. 1 O2 per NO), which is consistent with NO oxygenation. However, half-maximal rates of NO-stimulated O2 consumption required approx. 47µM O2; NO removal was ineffective at physiologically relevant O2 concentrations (below approx. 30µM O2). On exhaustion of O2, 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.
Escherichia coli flavohaemoglobin (Hmp) with equistoichiometric FAD and haem contents has a low affinity for dioxygen in the absence or presence of nitric oxide
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Catherine E. MILLS, Svetlana SEDELNIKOVA, Britta SØBALLE, Martin N. HUGHES, Robert K. POOLE; Escherichia coli flavohaemoglobin (Hmp) with equistoichiometric FAD and haem contents has a low affinity for dioxygen in the absence or presence of nitric oxide. Biochem J 15 January 2001; 353 (2): 207–213. doi: https://doi.org/10.1042/bj3530207
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