Escherichia coli flavohaemoglobin (Hmp) reduces cytochrome c and Fe(III)-hydroxamate K by electron transfer from NADH via FAD: sensitivity of oxidoreductase activity to haem-bound dioxygen

1 Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH, UK 2 Chemistry Department, King's College London, Strand, London WC2R 2LS, UK 3 Department 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 O 2 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 O 2 at the haem. The V max of cytochrome c reduction fell from 7.1 s -1 in the presence of 0.5 mM CO to 5.0 s -1 in the presence of 100 µM O 2 with no significant change in K m for cytochrome c (6.8 to 7.3 µM, respectively). O 2 at near-micromolar concentrations diminished cytochrome c reduction to a similar extent as did 100 µM O 2 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 O 2 since, in the absence of O 2 electron flux from FAD to other electron acceptors increases. However, the nature of such acceptors in vivo is not known and alternative models for O 2 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 Keywords: haemoglobin (bacterial), Escherichia coli , Hmp, NADH-cytochrome c oxidoreductase, oxygen sensor 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.