CO binding studies of nitric oxide synthase: effects of the substrate, inhibitors and tetrahydrobiopterin.

The dissociation constant (Kd) for CO from neuronal nitric oxide synthase heme in the absence of the substrate and cofactor was less than 10(-3) microM. In the presence of L-Arg, it dramatically increased up to 1 microM. In the presence of inhibitors such as N(G)-nitro-L-arginine methyl ester and 7-nitroindazole (NI), the Kd value further increased up to more than 100 microM. Addition of the cofactor, 5,6,7,8-tetrahydrobiopterin (H4B), increased the Kd value by 10-fold in the presence of L-Arg, whereas it decreased the value to less than one 250th in the presence of NI. Addition of H4B increased the recombination rate constant (k(on)) for CO by more than two-fold in the presence of L-Arg or N6-(1-iminoethyl)-L-lysine, whereas it decreased the k(on) value by three-fold in the presence of L-thiocitrulline. Thus, the binding fashion of some of inhibitors, such as NI, may be different from that of L-Arg with respect to the H4B effect.