Autoinhibition of neuronal nitric oxide synthase: distinct effects of reactive nitrogen and oxygen species on enzyme activity

Nitric oxide (NO) synthases (NOSs), which catalyse the oxidation of L-argin ine to L-citrulline and an oxide of nitrogen, possibly NO or nitroxyl (NO-) , are subject to autoinhibition by a mechanism that has yet to be fully elu cidated. In the present study we investigated the actions of NO and other N OS-derived products as possible autoregulators of enzyme activity. With the use of purified NOS-I, L-arginine turnover was found to operate initially at V-max (0-15 min, phase I) although, despite the presence of excess subst rate and cofactors, prolonged catalysis (15-90 min, phase II) was associate d with a rapid decline in L-arginine turnover. Taken together, these observ ations suggested that one or more NOS products inactivate NOS. Indeed, exog enously applied reactive nitrogen oxide species (RNSs) decreased V-max duri ng phase I, although with different potencies (NO- > NO > ONOO-); and effic acies (NO > NO- = ONOO-). The NO scavengers oxyhaemoglobin (HbO(2); 100 mu M) and 1H-imidazol-1-yloxy-2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramet hyl-3-oxide (CPTIO; 10 mu M) and the ONOO- scavenger GSH (7 mM) had no effe ct on NOS activity during phase I, except for an endogenous autoinhibitory influence of NO and ONOO-. However, superoxide dismutase (SOD; 300 units/ml ), which is thought either to increase the half-life of NO or to convert NO - to NO, lowered V-max in an NO-dependent manner because this effect was se lectively antagonized by HbO(2) (100 mu M). This latter observation demonst rated the requirement of SOD to reveal endogenous NO-mediated autoinhibitio n. Importantly, during phase II of catalysis, NOS became uncoupled and bega n to form H2O2 because catalase, which metabolizes H2O2, increased enzyme a ctivity. Consistent with this, exogenous H2O2 also inhibited NOS activity d uring phase I. Thus during catalysis NOS is subject to complex autoinhibiti on by both enzyme-derived RNS and H2O2, differentially affecting enzyme act ivity.