KINETICS OF CO LIGATION WITH NITRIC-OXIDE SYNTHASE BY FLASH-PHOTOLYSIS AND STOPPED-FLOW SPECTROPHOTOMETRY

Interaction of CO with hemeproteins has physiological importance. This is especially true for nitric-oxide synthases (NOS), heme/flavoenzyme s that produce (NO)-N-. and citrulline from L-arginine (Arg) and are i nhibited by CO in vitro. The kinetics of CO ligation with both neurona l NOS and its heme domain module were determined in the presence and a bsence of tetrahydrobiopterin and Arg to allow comparison with other h emeproteins. Geminate recombination in the nanosecond time domain is f ollowed by bimolecular association in the millisecond time domain. Com plex association kinetics imply considerable heterogeneity but can be approximated with two forms, one fast (2-3 x 10(6) M-1 s(-1)) and anot her slow (2-4 x 10(4) M-1 s(-1)). The relative proportions of the two forms vary with conditions. For the heme domain, fast forms dominate e xcept in the presence of both tetrahydrobiopterin and Arg, In the holo enzyme, slow forms dominate except when both reagents are absent. Gemi nate recombination is substantial, similar to 50%, only when fast form s predominate, Stopped-flow mixing found dissociation constants near 0 .3 s(-1). These data imply an equilibrium constant such that very litt le CO should bind at physiological conditions unless large CO concentr ations are present locally.