Regulation of inducible nitric oxide synthase by self-generated NO.
A ferric heme-nitric oxide (NO) complex can build up in mouse inducible nitric oxide synthase (iNOS) during NO synthesis from L-arginine. We investigated its formation kinetics, effect on catalytic activity, dependence on solution NO concentration, and effect on enzyme oxygen response (apparent KmO2). Heme-NO complex formation was biphasic and was linked kinetically to an inhibition of electron flux and catalysis in iNOS. Experiments that utilized a superoxide generating system to scavenge NO showed that the magnitude of heme-NO complex formation directly depended on the NO concentration achieved in the reaction solution. However, a minor portion of heme-NO complex (20%) still formed during NO synthesis even when solution NO was completely scavenged. Formation of the intrinsic heme-NO complex, and the heme-NO complex related to buildup of solution NO, increased the apparent KmO2 of iNOS 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 binding of solution NO, with the latter predominating when NO reaches high nanomolar to low micromolar concentrations. This behavior distinguishes iNOS from the other NOS isoforms and indicates a more complex regulation is possible for its activity and oxygen response in biologic settings.
Abu-Soud
H M
HM
Department of Immunology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio, 44195, USA. abusouh@ccf.org
Ichimori
K
K
Nakazawa
H
H
Stuehr
D J
DJ
D000818:Animals; D002384:Catalysis; D002417:Cattle; D005290:Ferric Compounds / Q000378:metabolism; D016166:Free Radical Scavengers / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010101:Oxygen Consumption; D012996:Solutions
D005290:Ferric Compounds; D016166:Free Radical Scavengers; D012996:Solutions; D009569:Nitric Oxide; D006418:Heme; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse