Calmodulin Activates Intersubunit Electron Transfer in the Neuronal Nitric-oxide Synthase Dimer

Koustubh Panda,Sanjay Ghosh,Dennis J Stuehr

doi:10.1074/jbc.m100687200

Abstract

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.

Highlights

C-terminal reductase domain that contains binding sites for FMN, FAD, and NADPH
The Neuronal nitric oxide synthase (nNOS) E592A mutation is analogous to the previously characterized E371A mutation in mouse inducible NO synthases (NOSs) (iNOS) and E361A mutation in human endothelial NOS (eNOS). Both mutations completely prevent Arg binding in homodimeric NOS [6, 44], and studies with iNOS show that a heterodimer containing only one E371A mutation displays normal Arg binding in the partner subunit that does not contain this mutation [34]
This is consistent with crystal structure data showing that Glu371 is a key protein residue that holds substrate Arg above the heme in the active site by forming hydrogen bonds between its carboxylate group and two guanidino nitrogens of Arg (9 –11). Because this Glu residue is highly conserved among all NOS isoforms [6, 44], we expected that its mutation would prevent Arg binding in nNOS

Summary

Electron Transfer Pathway in Neuronal NOS

35) to probe how CaM binding controls electron transfer in the heterodimer. The results clearly establish the path of electron transfer between flavin and heme groups in nNOS and reveal how CaM binding regulates heme reduction by the single reductase domain.

EXPERIMENTAL PROCEDURES

RESULTS

NADPH oxidation

DISCUSSION