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

Abstract

Cu2+ and Zn2+ inhibit all of the NADPH-dependent reactions catalyzed by neuronal nitric-oxide synthase (nNOS) including ferricytochrome c reduction, NADPH oxidation, and citrulline formation. Cu2+ and Zn2+ also inhibit ferricytochrome c reduction by the independent reductase domain. Zn2+ affects all activities of the full-length nNOS and the reductase domain to the same extent (estimated IC50 values from 9 to 31 μM), suggesting Zn2+ 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 Cu2+ with estimated IC50 values ranging from 6 to 33 μM. However, Cu2+ inhibits ferricytochrome c reduction by the full-length nNOS 2 orders of magnitude more potently, with an estimated IC50 value of 0.12 μM. These data suggest the possibility that Cu2+ may interact with nNOS at two sites, one composed exclusively of the reductase 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 ferricytochrome c reduction in full- length nNOS. Neither the inhibition by Cu2+ nor that by Zn2+ is dependent on calmodulin.