Cu2+ and Zn2+ inhibit nitric-oxide synthase through an interaction with the reductase domain

Cu2+ and Zn2+ inhibit all of the NADPH-dependent reactions catalyzed by neu ronal nitric-oxide synthase (nNOS) including ferricytochrome c reduction, N ADPH oxidation, and citrulline formation. Cu2+ and Zn2+ also inhibit ferric ytochrome c reduction by the independent reductase domain. Zn2+ affects all activities of the full-length nNOS and the reductase domain to the same ex tent (estimated IC50 values from 9 to 31 mu M), suggesting Zn2+ occupation of a single site in the reductase domain. Citrulline formation and NADPH ox idation by the full-length nNOS and ferricytochrome c reduction by the redu ctase domain are affected similarly by Cu2+, with estimated IC50 values ran ging from 6 to 33 mu M. However, Cu2+ inhibits ferricytochrome c reduction by the full-length nNOS 2 orders of magnitude more potently, with an estima ted IC50 value of 0.12 mu M. These data suggest the possibility that Cu2+ a my interact with nNOS at two sites, one composed exclusively of the reducta se domain (which is perhaps also involved in Zn2+-mediated inhibition), and another that includes components of both domains. Occupation of the second (higher affinity) site could then promote the selective inhibition of ferr icytochrome c reduction in full-length nNOS. Neither the inhibition by Cu2 nor that by Zn2+ is dependent on calmodulin.