ESCHERICHIA-COLI FLAVOHAEMOGLOBIN (HMP) REDUCES CYTOCHROME-C AND FE(III)-HYDROXAMATE-K BY ELECTRON-TRANSFER FROM NADH VIA FAD - SENSITIVITYOF OXIDOREDUCTASE ACTIVITY TO HEME-BOUND DIOXYGEN

Escherichia coil flavohaemoglobin (Hmp) reduced purified mitochondrial cytochrome c aerobically in a reaction that was not substantially inh ibited by superoxide dismutase, demonstrating that superoxide anion, t he product of O-2 reduction by Hmp, did not contribute markedly to cyt ochrome c reduction. Cytochrome c was reduced by Hmp even in the prese nce of 0.5 mRn 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 by. viciae anaerobi cally in a CO-insensitive manner, but at low rates and with low affini ty for this substrate. The NADH-cytochrome c oxidoreductase activity o f Hmp was slightly sensitive to the binding and reduction of O-2 at th e 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 mu M O-2, w ith no significant change in K-m for cytochrome c (6.8 to 7.3 mu M, re spectively). O-2 at near-micromolar concentrations diminished cytochro me c reduction to a similar extent as did 100 mu M O-2. Thus, Hmp acts as a reductase of broad specificity, apparently without involvement o f electron transfer via the globin-like haem. These data are consisten t 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 ele ctron accepters increases. However, the nature of such accepters in vi vo is not known and alternative models for O-2 sensing are also consid ered.