The 42-amino acid insert in the FMN domain of neuronal nitric-oxide synthase exerts control over Ca2+/calmodulin-dependent electron transfer

The neuronal and endothelial nitric-oxide synthases (nNOS and eNOS) differ from inducible NOS in their dependence on the intracellular Ca2+ concentration. Both nNOS and eNOS are activated by the reversible binding of calmodulin (CaM) in the presence of Ca2+, whereas inducible NOS binds CaM irreversibly. One major divergence in the close sequence similarity between the NOS isoforms is a 40-50-amino acid insert in the middle of the FMN-binding domains of nNOS and eNOS. It has previously been proposed that this insert forms an autoinhibitory domain designed to destabilize CaM binding and increase its Ca2+ dependence. To examine the importance of the insert we constructed two deletion mutants designed to remove the bulk of it from nNOS. Both mutants (Delta 40 and Delta 42) retained maximal NO synthesis activity at lower concentrations of free Ca2+ than the wild type enzyme. They were also found to retain 30% of their activity in the absence of Ca2+/CaM, indicating that the insert plays an important role in disabling the enzyme when the physiological Ca2+ concentration is low. Reduction of nNOS heme by NADPH under rigorous anaerobic conditions was found to occur in the wild type enzyme only in the presence of Ca2+/CaM. However, reduction of heme in the Delta 40 mutant occurred spontaneously on addition of NADPH in the absence of Ca2+/CaM. This suggests that the insert regulates activity by inhibiting electron transfer from FMN to heme in the absence of Ca2+/CaM and by destabilizing CaM binding at low Ca2+ concentrations, consistent with its role as an autoinhibitory domain.