REACTIONS OF THE ESCHERICHIA-COLI FLAVOHAEMOGLOBIN (HMP) WITH OXYGEN AND REDUCED NICOTINAMIDE ADENINE-DINUCLEOTIDE - EVIDENCE FOR OXYGEN SWITCHING OF FLAVIN OXIDOREDUCTION AND A MECHANISM FOR OXYGEN SENSING

The soluble flavohaemoglobin (Hmp) of Escherichia coli contains haem B and FAD in a single 44 kDa polypeptide, and shows NADH oxidase activi ty. The oxidized protein reacted rapidly with NADH in the presence of 0, to form an oxygenated species while the flavin remained largely oxi dized. Spectral and kinetic analyses revealed rapid biphasic reduction and oxygenation of high-spin haem with apparent relaxation times of 6 and 64 ms at pH 8 and 25-degrees-C, suggestive of a significant physi ological role for the protein. This was followed by a monophasic reduc tion of the flavin with a relaxation time of 92 ms. On exhaustion of o xygen, the oxygenated haem was converted into the deoxy form biphasica lly with relaxation times of 43 and 170 s, followed by extensive reduc tion of the flavin with corresponding relaxation times of 70 and 256 s . Based on these observations, we propose that Hmp could act as an oxy gen sensor in E. coli by combining with intracellular oxygen, thus lim iting flavin reduction in the aerobic steady state. Lowering of the ox ygen concentration causes dissociation of the oxy species and sustaine d flavin reduction. Because Hmp can reduce Fe(III), such a mechanism m ight control, for example, flavin-mediated Fe(III) reduction required for activation of the anaerobic gene regulator, Fnr.