Lys842 in Neuronal Nitric-oxide Synthase Enables the Autoinhibitory Insert to Antagonize Calmodulin Binding, Increase FMN Shielding, and Suppress Interflavin Electron Transfer

Zhi-Wen Guan,Dennis J. Stuehr,Mohammad Mahfuzul Haque,Elizabeth D. Getzoff,Chin-Chuan Wei,Elsa D. Garcin

doi:10.1074/jbc.m109.000810

Abstract

Neuronal nitric-oxide synthase (nNOS) contains a unique autoinhibitory insert (AI) in its FMN subdomain that represses nNOS reductase activities and controls the calcium sensitivity of calmodulin (CaM) binding to nNOS. How the AI does this is unclear. A conserved charged residue (Lys(842)) lies within a putative CaM binding helix in the middle of the AI. We investigated its role by substituting residues that neutralize (Ala) or reverse (Glu) the charge at Lys(842). Compared with wild type nNOS, the mutant enzymes had greater cytochrome c reductase and NADPH oxidase activities in the CaM-free state, were able to bind CaM at lower calcium concentration, and had lower rates of heme reduction and NO synthesis in one case (K842A). Moreover, stopped-flow spectrophotometric experiments with the nNOS reductase domain indicate that the CaM-free mutants had faster flavin reduction kinetics and had less shielding of their FMN subdomains compared with wild type and no longer increased their level of FMN shielding in response to NADPH binding. Thus, Lys(842) is critical for the known functions of the AI and also enables two additional functions of the AI as newly identified here: suppression of electron transfer to FMN and control of the conformational equilibrium of the nNOS reductase domain. Its effect on the conformational equilibrium probably explains suppression of catalysis by the AI.

Highlights

Are composed of an N-terminal oxygenase domain that contains (6R)-tetrahydrobiopterin, heme, and an L-arginine binding site and a C-terminal reductase domain that contains FAD, FMN, and an NADPH binding site (4 –10), with the domains being linked together by a central calmodulin (CaM)-binding element (4, 8)
Steady-state Catalytic Activities—Table 1 lists the nitric oxide (NO) synthesis, NADPH oxidation, cytochrome c reductase, and ferricyanide reductase activities we obtained for Neuronal nitric-oxide synthase (nNOS) and the Lys842 mutants
The autoinhibitory insert (AI) is unique to the nitric-oxide synthases (NOSs) family of flavoproteins, and its mechanisms of action are of current interest

Summary

EXPERIMENTAL PROCEDURES

Reactions were initiated by rapid mixing an anaerobic CO-saturated solution containing 50 ␮M NADPH with an anaerobic CO-saturated solution containing 5 ␮M of WT or mutant nNOS in 40 mM EPPS buffer, pH 7.6, containing 10 ␮M (6R)-tetrahydrobiopterin, 1 mM L-arginine, 10 ␮M CaM, and 1 mM Ca2ϩ. The percentage of flavin reduction that takes place in the instrument mixing dead time was calculated after obtaining absorbance traces from control stopped-flow reactions that rapidly mixed the same enzyme solutions described above with buffer that did not contain NADPH, as previously described in detail (37). Anaerobic Pre-steady-state Cytochrome c Reduction—The rate of cytochrome c reduction by an excess of prereduced nNOSred proteins under various conditions was measured in the stopped-flow apparatus (SF-51MX, HiTech Ltd.) at 10 °C as described previously (37). An initial concentration of 20 ␮M Ca2ϩ was added into each of the reaction mixtures prior to the EGTA titration

RESULTS

42 Ϯ 3 54 Ϯ 5

DISCUSSION