REACTIONS OF THE ESCHERICHIA-COLI FLAVOHAEMOGLOBIN (HMP) WITH NADH AND NEAR-MICROMOLAR OXYGEN - OXYGEN-AFFINITY OF NADH OXIDASE ACTIVITY

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.