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. H(2)O(2) accumulated on NADH oxidation by the purified protein and in cell extracts with elevated Hmp contents. H(2)O(2) 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 microM; the addition of 15 microM FAD decreased the K(m) for oxygen to 15 microM without a change in V(max) but catalysed cyanide-insensitive oxygen consumption, attributed to electron transfer from flavins 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 microM O(2); NO removal was ineffective at physiologically relevant O(2) concentrations (below approx. 30 microM 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.
Mills
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CE
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Sheffield, S10 2TN, U.K.
Sedelnikova
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S
Søballe
B
B
Hughes
M N
MN
Poole
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RK
D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004093:Dihydropteridine Reductase; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000494:pharmacology; D006420:Hemeproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases; D009569:Nitric Oxide / Q000494:pharmacology*; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D010101:Oxygen Consumption
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D005182:Flavin-Adenine Dinucleotide; D009569:Nitric Oxide; D004093:Dihydropteridine Reductase; C076538:hmp protein, E coli; D009247:NADH, NADPH Oxidoreductases; D010100:Oxygen