Cu(2+) and Zn(2+) inhibit all of the NADPH-dependent reactions catalyzed by neuronal nitric-oxide synthase (nNOS) including ferricytochrome c reduction, NADPH oxidation, and citrulline formation. Cu(2+) and Zn(2+) also inhibit ferricytochrome c reduction by the independent reductase domain. Zn(2+) affects all activities of the full-length nNOS and the reductase domain to the same extent (estimated IC(50) values from 9 to 31 microm), suggesting Zn(2+) occupation of a single site in the reductase domain. Citrulline formation and NADPH oxidation by the full-length nNOS and ferricytochrome c reduction by the reductase domain are affected similarly by Cu(2+), with estimated IC(50) values ranging from 6 to 33 microm. However, Cu(2+) inhibits ferricytochrome c reduction by the full-length nNOS 2 orders of magnitude more potently, with an estimated IC(50) value of 0.12 microm. These data suggest the possibility that Cu(2+) may interact with nNOS at two sites, one composed exclusively of the reductase domain (which is perhaps also involved in Zn(2+)-mediated inhibition), and another that includes components of both domains. Occupation of the second (higher affinity) site could then promote the selective inhibition of ferricytochrome c reduction in full-length nNOS. Neither the inhibition by Cu(2+) nor that by Zn(2+) is dependent on calmodulin.