Domain interactions in neuronal nitric oxide synthase identified by time-resolved fluorescence

Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide through the transfer of electrons from FAD to FMN in the reductase domain of the enzyme and then from FMN to a heme in the oxygenase domain of the partner enzyme in a homodimeric complex. The calcium-signaling protein calmodulin (CaM) binds to a CaM-binding domain located between the reductase and oxygenase domains to enable efficient NOS activity. Directed electron transfer in NOS occurs through the formation of conformational states of the enzyme that sequentially place electron donor and acceptor domains nearby, suggesting that the electron transfers are conformationally gated. We used fluorescence-labeled CaM and time-resolved fluorescence detection to reveal the presence of multiple conformational states of NOS. The efficiency of FRET from a fluorophore attached to CaM to the heme groups of the enzyme depends on the conformational state of the enzyme. Analysis of fluorescence decays for a series of site-directed mutants of NOS and CaM allowed us to assign fluorescence quenching states to conformational states of the enzyme. We used mutations expected to disrupt specific interactions between enzyme domains of NOS or between CaM and NOS, consistent with their known effects on enzyme kinetics. The results suggest the presence of a conformational state in which the FAD and FMN subdomains are in close proximity, one or more in which the FMN subdomain interacts with the oxygenase domain in the partner enzyme, and multiple conformation states in which CaM is in close proximity to one of the oxygenase domains. We suggest that CaM docking to the oxygenase domain facilitates the productive interaction of the FMN domain with the oxygenase domain in its dimeric partner. Such a conformation would enable electron transfer from FMN to the heme of the dimeric partner.