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1Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH, UK
2Chemistry Department, King's College London, Strand, London WC2R 2LS, UK
3Department of Public Health, Graduate School of Medicine, Kyoto University, Kyoto 606, Japan
ABSTRACT
Escherichia coli flavohaemoglobin (Hmp) reduced purified mitochondrial cytochrome c aerobically in a reaction that was not substantially inhibited by superoxide dismutase, demonstrating that superoxide anion, the product of O2 reduction by Hmp, did not contribute markedly to cytochrome c reduction. Cytochrome c was reduced by Hmp even in the presence of 0.5 mM CO, when the haem B was locked in the ferrous, low-spin state, demonstrating that electron transfer to cytochrome c from NADH was via FAD, not haem. Hmp also reduced the ferrisiderophore complex Fe(III)-hydroxamate K from Rhizobium leguminosarum bv. viciae anaerobically in a CO-insensitive manner, but at low rates and with low affinity for this substrate. The NADH-cytochrome c oxidoreductase activity of Hmp was slightly sensitive to the binding and reduction of O2 at the haem. The Vmax of cytochrome c reduction fell from 7.1 s-1in the presence of 0.5 mM CO to 5.0 s-1in the presence of 100 µM O2with no significant change in Km for cytochrome c (6.8 to 7.3 µM, respectively). O2 at near-micromolar concentrations diminished cytochrome c reduction to a similar extent as did 100 µM O2 Thus, Hmp acts as a reductase of broad specificity, apparently without involvement of electron transfer via the globin-like haem. These data are consistent with the hypothesis that Hmp could act as an intracellular sensor of O2 since, in the absence of O2 electron flux from FAD to other electron acceptors increases. However, the nature of such acceptors in vivo is not known and alternative models for O2 sensing are also considered.
Author for correspondence: Robert K. Poole. Tel: +44 114 222 4447. Fax: +44 114 272 8697. e-mail: r.poole@sheffield.ac.uk
Present address: Department of Paediatrics, Imperial College School of Medicine at St Mary's, London W2 1PG, UK.
Present address: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
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