Recent studies have uncovered several interesting and unusual mechanisms that control electron transfer within neuronal nitric oxide synthase (NOS). Each subunit of neuronal NOS is composed of a reductase domain that contains the binding sites for NADPH and flavins and an oxygenase domain that contains the binding sites for heme, L-arginine, and H4biopterin. A calmodulin (CaM) binding site is located between the domains. Electrons provided by NADPH are transferred in a linear sequence, first entering the NOS flavins and then passing across the domains to the heme iron. Reduction of the NOS heme iron enables it to bind oxygen and catalyze nitric oxide (NO) synthesis. CaM plays a role in mediating the flavin-to-heme interdomain electron transfer. Electrons from NADPH load into the flavins of CaM-free neuronal NOS, but can transfer to the NOS heme iron only when CaM binds. The CaM-triggered electron transfer to heme iron occurs independently of substrate binding, causes rapid enzymatic oxidation of NADPH in the presence of O2, and is required in order for NO synthesis to occur. CaM binding to NOS is associated with an increase in enzyme tryptophan and flavin fluorescence, suggesting that NOS structural changes induced by CaM may trigger the interdomain electron transfer. CaM-binding neither affects NOS affinity for its substrate L-arginine, nor does it alter the ability of the NOS heme iron to bind ligands such as cyanide or CO. The ability of CaM to trigger interdomain electron transfer within a redox enzyme is novel and therefore reveals a new function for calcium-binding proteins in biology.
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