Regulation of inducible nitric oxide synthase by self-generated NO

A ferric heme-nitric oxide (NO) complex can build up in mouse inducible nit ric oxide synthase (iNOS) during NO synthesis from L-arginine. We investiga ted its formation kinetics, effect on catalytic activity, dependence on sol ution NO concentration, and effect on enzyme oxygen response (apparent KmO( 2)). Heme-NO complex formation was biphasic and was linked kinetically to a n inhibition of electron flux and catalysis in iNOS. Experiments that utili zed a superoxide generating system to scavenge NO showed that the magnitude of heme-NO complex formation directly depended on the NO concentration ach ieved in the reaction solution. However, a minor portion of heme-NO complex (20%) still formed during NO synthesis even when solution NO was completel y scavenged. Formation of the intrinsic heme-NO complex, and the heme-NO co mplex related to buildup of solution NO, increased the apparent KmO2 of iNO S by 10- and 4-fold, respectively. Together, the data show heme-NO complex buildup in iNOS is due to both intrinsic NO binding and to equilibrium bind ing of solution NO, with the latter predominating when NO reaches high nano molar to low micromolar concentrations. This behavior distinguishes iNOS fr om the other NOS isoforms and indicates a more complex regulation is possib le for its activity and oxygen response in biologic settings.