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 O₂ with nitric oxide (NO) to form nitrate [HMP-Fe(II)O₂ + NO → HMP-Fe(III) + NO₃ ^-] and, under anaerobic conditions, a slower reduction of heme-bound NO to an NO^- equivalent (followed by the formation of N₂O), thereby protecting against nitrosative stress under both aerobic and anaerobic conditions, and rationalizing our finding that NO is rapidly consumed across a wide range of O₂ concentrations. It has been alternatively suggested that HMP activity is inhibited at low pO₂ because the enzyme is then in the relatively inactive nitrosyl form [k_off/k_on for NO (0.000008 μM) ≪ k_off/k_on for O₂ (0.012 μM) and K_M for O₂ = 30-100 μM]. To resolve this discrepancy, we have directly measured heme-ligand turnover and NADH consumption under various O₂/NO concentrations. We find that, at biologically relevant O₂ concentrations, HMP preferentially binds NO (not O₂), which it then reacts with oxygen to form nitrate (in essence NO^- + O₂ → NO₃ ^-). During steady-state turnover, the enzyme can be found in the ferric (FeIII) state. The formation of a heme-bound nitroxyl equivalent and its subsequent oxidation is a novel enzymatic function, and one that dominates the oxygenase activity under biologically relevant conditions. These data unify the mechanism of HMP/NO interaction with those recently described for the nematode Ascaris and mammalian hemoglobins, and more generally suggest that the peroxidase (FeIII)-like properties of globins have evolved for handling of NO.