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 nitrate [HMP-Fe(II)O 2 + NO → HMP-Fe(III) + NO [12pt]{minimal} {amsmath} {wasysym} {amsfonts} {amssymb} {amsbsy} {mathrsfs} {}{-69pt} {document} {equation*}{{₃⁻}}{equation*}{document} ] and, under anaerobic conditions, a slower reduction of heme-bound NO to an NO − equivalent (followed by the formation of N 2 O), 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 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 μM) ≪ k off / k on for O 2 (0.012 μM) and K M for O 2 = 30–100 μM]. To resolve this discrepancy, we have directly measured heme-ligand turnover and NADH consumption under various O 2 /NO concentrations. We find that, at biologically relevant O 2 concentrations, HMP preferentially binds NO (not O 2 ), which it then reacts with oxygen to form nitrate (in essence NO − + O 2 → NO [12pt]{minimal} {amsmath} {wasysym} {amsfonts} {amssymb} {amsbsy} {mathrsfs} {}{-69pt} {document} {equation*}{{₃⁻}}{equation*}{document} ). 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.