L-ARGININE AND CALMODULIN REGULATION OF THE HEME IRON REACTIVITY IN NEURONAL NITRIC-OXIDE SYNTHASE

Neuronal nitric oxide synthase (NOS) is a calmodulin-dependent, flavin -containing hemoprotein that forms NO from L-arginine, NADPH, and mole cular oxygen. Calmodulin binding to NOS triggers reduction of its heme groups (Abu-Soud, H., and Stuehr, D. J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10769-10762), leading to NADPH oxidation and NO synthesis. We have examined how L-arginine and calmodulin control the ligand bin ding and electron acceptor properties of the NOS heme iron. In the abs ence of bound calmodulin, ferric NOS exhibited a K-d of 0.6 mu M for L -arginine, as determined by the substrate-dependent shift in heme spin equilibrium toward a high spin state. L-Arginine binding reduced the affinity of the ferric NOS heme for cyanide by 8-fold. Carbon monoxide binding to substrate-free ferrous NOS occurred at a rate of 2 x 10(5) M(-1) s(-1); this rate was decreased 12-fold when L-arginine was boun d. In contrast, bound calmodulin did not significantly affect cyanide or carbon monoxide binding to the NOS heme, nor did it alter NOS bindi ng affinity for L-arginine. Anaerobic titration of a calmodulin-bound, L-arginine-free NOS with NADPH led to incomplete reduction of the hem e iron; full reduction was achieved only in the presence of added L-ar ginine. Thus, our data suggest that L-arginine controls NOS heme iron reactivity in at least two ways: 1) it slows ligand interactions by bi nding in the distal pocket very near the heme and 2) it also appears t o increase the reduction potential of the iron. In contrast, bound cal modulin does not alter the NOS affinity for L-arginine or heme ligands and may function solely as a switch that enables electrons to pass fr om the flavin domain onto the heme iron.