Rk. Poole et al., REACTIONS OF THE ESCHERICHIA-COLI FLAVOHAEMOGLOBIN (HMP) WITH NADH AND NEAR-MICROMOLAR OXYGEN - OXYGEN-AFFINITY OF NADH OXIDASE ACTIVITY, Microbiology, 142, 1996, pp. 1141-1148
The soluble flavohaemoglobin (Hmp) of Escherichia coli, product of the
hmp gene, contains haem B and FAD in a single polypeptide of molecula
r mass 44 kDa, The function of this protein (and of the similar protei
ns identified in several bacteria and yeast) is unknown, but the obser
vation that the binding of oxygen to haem modulates the reduction leve
l of FAD has suggested that Hmp could act as an oxygen sensor. Here, s
topped-flow, rapid-scan spectroscopy has shown that the oxidized prote
in reacts rapidly with NADH to form an oxygenated species, even when e
fforts are made to reduce oxygen concentrations to sub-micromolar leve
ls, suggesting a high affinity for this ligand. As is the case at high
oxygen concentrations (130 mu M), oxygenated species formation was ki
netically and spectrally heterogeneous. Between 12 ms and 1 s after mi
xing, following transient formation of the deoxy form and its reaction
with dioxygen, a steady-state level of the oxygenated species was att
ained. During the oxygenated steady state, the flavin remained largely
oxidized, as observed previously at 130 mu M oxygen. Hmp is an NADH o
xidase; on exhaustion of oxygen by reduction (in <10 s under these con
ditions), the oxygenated species disappeared to generate the deoxy Fe(
II) haem, whereupon the flavin was reduced. The affinity for oxygen du
ring NADH oxidation was measured by continuous dual-wavelength monitor
ing of the deoxygenation of oxymyoglobin. The K-m for oxygen was 2.6 m
u M, much higher than the K-m values determined, using the same method
, for tire membrane-bound terminal oxidases cytochromes bo' and bd. Th
ese results show that the oxidase activity of Hmp, but not necessarily
oxygen binding, would be minimal at oxygen concentrations that limit
terminal oxidase function.