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Volume 142, Issue 5

Reactions of the Escherichia coli flavohaemoglobin (Hmp) with NADH and near-micromolar oxygen: oxygen affinity of NADH oxidase activity Free

Abstract

The soluble flavohaemoglobin (Hmp) of , product of the gene, contains haem B and FAD in a single polypeptide of molecular mass 44 kDa. The function of this protein (and of the similar proteins identified in several bacteria and yeast) is unknown, but the observation that the binding of oxygen to haem modulates the reduction level of FAD has suggested that Hmp could act as an oxygen sensor. Here, stopped-flow, rapid-scan spectroscopy has shown that the oxidized protein reacts rapidly with NADH to form an oxygenated species, even when efforts are made to reduce oxygen concentrations to sub-micromolar levels, suggesting a high affinity for this ligand. As is the case at high oxygen concentrations (130 μM), oxygenated species formation was kinetically and spectrally heterogeneous. Between 12 ms and 1 s after mixing, following transient formation of the deoxy form and its reaction with dioxygen, a steady-state level of the oxygenated species was attained. During the oxygenated steady state, the flavin remained largely oxidized, as observed previously at 130 μM oxygen. Hmp is an NADH oxidase; on exhaustion of oxygen by reduction (in < 10 s under these conditions), the oxygenated species disappeared to generate the deoxy Fe(II) haem, whereupon the flavin was reduced. The affinity for oxygen during NADH oxidation was measured by continuous dual-wavelength monitoring of the deoxygenation of oxymyoglobin. The for oxygen was 2.6 μM, much higher than the values determined, using the same method, for the membrane-bound terminal oxidases cytochromes and . These results show that the oxidase activity of Hmp, but not necessarily oxygen binding, would be minimal at oxygen concentrations that limit terminal oxidase function.

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Keyword(s): Escherichia coli , haemoglobin (bacterial) , Hmp, oxygen affinity and oxidase
© Society for General Microbiology 1996
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1996-05-01
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Reactions of the Escherichia coli flavohaemoglobin (Hmp) with NADH and near-micromolar oxygen: oxygen affinity of NADH oxidase activity
Microbiology 142, 1141 (1996); https://doi.org/10.1099/13500872-142-5-1141
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