Electron Transfer by Neuronal Nitric-oxide Synthase Is Regulated by Concerted Interaction of Calmodulin and Two Intrinsic Regulatory Elements

Linda J. Roman,Bettie Sue S. Masters

doi:10.1074/jbc.m603671200

Abstract

The nitric-oxide synthases (NOSs) are modular, cofactor-containing enzymes, divided into a heme-containing oxygenase domain and an FMN- and FAD-containing reductase domain. The domains are connected by a calmodulin (CaM)-binding sequence, occupancy of which is required for nitric oxide (NO) production. Two additional CaM-modulated regulatory elements are present in the reductase domains of the constitutive isoforms, the autoregulatory region (AR) and the C-terminal tail region. Deletion of the AR reduces CaM stimulation of electron flow through the reductase domain from 10-fold in wild-type nNOS to 2-fold in the mutant. Deletion of the C terminus yields an enzyme with greatly enhanced reductase activity in the absence of CaM but with activity equivalent to that of wild-type enzyme in its presence. A mutant in which both the AR and C terminus were deleted completely loses CaM modulation through the reductase domain. Thus, transduction of the CaM effect through the reductase domain of nNOS is dependent on these elements. Formation of nitric oxide is, however, still stimulated by CaM in all three mutants. A CaM molecule in which the N-terminal lobe was replaced by the C-terminal lobe (CaM-CC) supported NO synthesis by the deletion mutants but not by wild-type nNOS. We propose a model in which the AR, the C-terminal tail, and CaM interact directly to regulate the conformational state of the reductase domain of nNOS.

Highlights

The immune response, depending on the cell type in which it is being expressed
All three nitric-oxide synthases (NOSs) isoforms are modular, cofactor-containing enzymes. They can be roughly divided into an oxygenase domain, containing heme, tetrahydrobiopterin, and the arginine-binding site, and a reductase domain, containing the flavins FMN and FAD, as well as the NADPH-binding site. These two domains are connected by a calmodulin (CaM)-binding site, which is always occupied under physiological conditions by CaM in iNOS, whereas CaM binding to nNOS and eNOS requires an increase in intracellular calcium
Recent models [12, 13] propose that bound NADPH locks the enzyme into a state in which the FMN domain is closely associated with the FAD and NADPH binding domains, and the FMN is deeply buried in the protein, inaccessible to other electron acceptors

Summary

EXPERIMENTAL PROCEDURES

Chemicals—(6R)-5,6,7,8-Tetrahydrobiopterin was from Research Biochemicals International (Natick, MA). Ferricyanide reduction was performed in the same man- deleted, CaM modulation of reductase activity is decreased but ner as cytochrome c reduction except that the ferricyanide con- still evident; nNOS-AR is stimulated only about 2-fold by centration was varied and the extinction coefficient was 1.02 CaM When both the autoregulatory region and the C-termMϪ1 cmϪ1 at 420 nm. The reoxidation of reduction, indicating CaM modulation of electron flow reduced flavins was monitored at 485 nm for all enzymes in the through the reductase domain is implemented entirely by presence of 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 20 ␮M these two regulatory elements. NADPH, and 2 ␮M enzyme at 23 °C [19, 20]

RESULTS

Construct kcat

Findings

DISCUSSION