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
Rk. Poole et al., 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, Microbiology, 143, 1997, pp. 1557-1565
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.