Calmodulin activates intersubunit electron transfer in the neuronal nitric-oxide synthase dimer

Neuronal nitric oxide synthase (nNOS) is composed of an oxygenase domain that binds heme, (6R)-tetrahydrobiopterin, and Arg, coupled to a reductase domain that binds FAD, FMN, and NADPH. Activity requires dimeric interaction between two oxygenase domains and calmodulin binding between the reductase and oxygenase domains, which triggers electron transfer between flavin and heme groups. We constructed four different nNOS heterodimers to determine the path of calmodulin-induced electron transfer in a nNOS dimer, A predominantly monomeric mutant of rat nNOS (G671A) and its Arg binding mutant (G671A/E592A) were used as full-length subunits, along with oxygenase domain partners that either did or did not contain the E592A mutation. The E592A mutation prevented Arg binding to the oxygenase domain in which it was present. It also prevented NO synthesis when it was located in the oxygenase domain adjacent to the full-length subunit, However, it had no effect when present in the full-length subunit (i.e. the subunit containing the reductase domain). The active heterodimer (G671A/E592A full-length subunit plus wild type oxygenase domain subunit) showed remarkable similarity with wild type homodimeric nNOS in its catalytic responses to five different forms and chimeras of calmodulin, This reveals an active involvement of calmodulin in supporting transelectron transfer between flavin and heme groups on adjacent subunits in nNOS, In summary, we propose that calmodulin functions to properly align adjacent reductase and the oxygenase domains in a nNOS dimer for electron transfer between them, leading to NO synthesis by the heme.