Flavohemoglobin denitrosylase catalyzes the reaction of a nitroxyl equivalent with molecular oxygen

We have previously reported that bacterial flavohemoglobin (HMP) catalyzes both a rapid reaction of heme-bound O-2. with nitric oxide (NO) to form nit rate [HMP-Fe(II)O-2 + NO HMP-Fe(III) + NO3-] and, under anaerobic condition s, a slower reduction of heme-bound NO to an NO- equivalent (followed by th e formation of N2O), thereby protecting against nitrosative stress under bo th aerobic and anaerobic conditions, and rationalizing our finding that NO is rapidly consumed across a wide range Of O-2 concentrations. It has been alternatively suggested that HMP activity is inhibited at low pO(2) because the enzyme is then in the relatively inactive nitrosyl form [k(off)/k(on) for NO (0.000008 muM)) much less than k(off)/k(on) for O-2 (0.012 muM) and K-M for O-2 = 30-100 muM]. To resolve this discrepancy, we have directly me asured heme-ligand turnover and NADH consumption under various 02/NO concen trations. We find that, at biologically relevant O-2 concentrations, HMP pr eferentially binds NO (not O-2), which it then reacts with oxygen to form n itrate (in essence NO- + O-2 --> NO3-. During steady-state turnover, the en zyme can be found in the ferric (FeIII) state. The formation of a heme-boun d nitroxyl equivalent and its subsequent oxidation is a novel enzymatic fun ction, and one that dominates the oxygenase activity under biologically rel evant conditions. These data unify the mechanism of HMP/NO interaction with those recently described for the nematode Ascaris and mammalian hemoglobin s, and more generally suggest that the peroxidase (FeIII)-like properties o f globins have evolved for handling of NO.