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 molecular 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 binding and electron acceptor properties of the NOS heme iron. In the absence of bound calmodulin, ferric NOS exhibited a Kd of 0.6 microM 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 bound. In contrast, bound calmodulin did not significantly affect cyanide or carbon monoxide binding to the NOS heme, nor did it alter NOS binding affinity for L-arginine. Anaerobic titration of a calmodulin-bound, L-arginine-free NOS with NADPH led to incomplete reduction of the heme iron; full reduction was achieved only in the presence of added L-arginine. Thus, our data suggest that L-arginine controls NOS heme iron reactivity in at least two ways: 1) it slows ligand interactions by binding in the distal pocket very near the heme and 2) it also appears to increase the reduction potential of the iron. In contrast, bound calmodulin does not alter the NOS affinity for L-arginine or heme ligands and may function solely as a switch that enables electrons to pass from the flavin domain onto the heme iron.
Matsuoka
A
A
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970.
Stuehr
D J
DJ
Olson
J S
JS
Clark
P
P
Ikeda-Saito
M
M
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D001120:Arginine / Q000502:physiology*; D001665:Binding Sites; D002147:Calmodulin / Q000502:physiology*; D002248:Carbon Monoxide / Q000378:metabolism; D002478:Cells, Cultured; D006418:Heme / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D007501:Iron / Q000032:analysis / Q000502:physiology*; D007700:Kinetics; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase
D002147:Calmodulin; D006418:Heme; D002248:Carbon Monoxide; D001120:Arginine; D007501:Iron; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases