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.
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-1 in the presence of 0.5 mM CO to 5.0 s-1 in the presence of 100 microM O2, with no significant change in K(m) for cytochrome c (6.8 to 7.3 microM, respectively). O2 at near-micromolar concentrations diminished cytochrome c reduction to a similar extent as did 100 microM 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.
Poole
Robert K
RK
Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH, UK.
Rogers
Nicola J
NJ
Chemistry Department, King's College London, Strand, London WC2R 2LS, UK.|Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH, UK.
D'mello
Rita A M
RAM
Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH, UK.
Hughes
Martin N
MN
Chemistry Department, King's College London, Strand, London WC2R 2LS, UK.
Orii
Yutaka
Y
Department of Public Health, Graduate School of Medicine, Kyoto University, Kyoto 606, Japan.
D000332:Aerobiosis; D000693:Anaerobiosis; D002248:Carbon Monoxide / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D004579:Electron Transport; D004926:Escherichia coli / Q000378:metabolism*; D005290:Ferric Compounds / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006877:Hydroxamic Acids / Q000378:metabolism*; D007700:Kinetics; D024101:Mitochondrial Proteins; D009124:Muscle Proteins / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology*; D016961:Rhizobium leguminosarum / Q000378:metabolism; D013379:Substrate Specificity; D013482:Superoxide Dismutase / Q000494:pharmacology
D003574:Cytochrome c Group; D005290:Ferric Compounds; D006877:Hydroxamic Acids; C088548:IMMT protein, human; D024101:Mitochondrial Proteins; D009124:Muscle Proteins; C074579:iron (III) hydroxamate; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D002248:Carbon Monoxide; D013482:Superoxide Dismutase; D010100:Oxygen