Interaction between caveolin-1 and the reductase domain of endothelial nitric-oxide synthase. Consequences for catalysis

Endothelial nitric-oxide synthase (eNOS) is targeted to caveoli through interaction with caveolin-1 (cav-1). cav-1 binding to a consensus site in the eNOS oxygenase domain is proposed to antagonize calmodulin (CaM) binding and thereby inhibit eNOS nitric oxide (NO) synthesis. To study the mechanism, we examined how cav-1 scaffolding domain peptide (amino acids 82-101; cav-1P) would affect NO synthesis, NADPH oxidation, cytochrome c reduction, and ferricyanide reduction by full-length eNOS or its isolated oxygenase and reductase domains. Cav-1P equivalently inhibited NO synthesis and NADPH oxidation by full-length eNOS in a manner reversible by CaM but did not affect NADPH-independent NO synthesis by full-length eNOS or its oxygenase domain, indicating inhibition required the reductase domain. Similar concentrations of cav-1P inhibited cytochrome c reduction by full-length eNOS or the reductase domain (amino acids 492-1205) in a CaM-reversible manner, indicating cav-1P interaction with reductase or full-length eNOS are equivalent. Ferricyanide reduction was unaffected by cav-1P in all cases. Immunoblotting showed that full-length eNOS, eNOS oxygenase, and eNOS reductase all bound to an immobilized glutathione S-transferase-cav-1 fusion protein. Thus, cav-1 interacts independently with both oxygenase and reductase domains of eNOS. The reductase interaction occurs independent of a cav-1 binding motif, is CaM-reversible, and is of sufficient affinity to match cav-1P inhibition of NO synthesis by full-length eNOS. We propose that cav-1 binding to eNOS reductase compromises its ability to bind CaM and to donate electrons to the eNOS heme, thereby inhibiting NO synthesis.