Differences in a Conformational Equilibrium Distinguish Catalysis by the Endothelial and Neuronal Nitric-oxide Synthase Flavoproteins

Dennis J. Stuehr,Russ Hille,Mauro Tiso,Deborah Durra,David W. Konas,Craig Hemann,Robielyn P. Ilagan

doi:10.1074/jbc.m802914200

Abstract

Nitric oxide (NO) is a physiological mediator synthesized by NO synthases (NOS). Despite their structural similarity, endothelial NOS (eNOS) has a 6-fold lower NO synthesis activity and 6-16-fold lower cytochrome c reductase activity than neuronal NOS (nNOS), implying significantly different electron transfer capacities. We utilized purified reductase domain constructs of either enzyme (bovine eNOSr and rat nNOSr) to investigate the following three mechanisms that may control their electron transfer: (i) the set point and control of a two-state conformational equilibrium of their FMN subdomains; (ii) the flavin midpoint reduction potentials; and (iii) the kinetics of NOSr-NADP+ interactions. Although eNOSr and nNOSr differed in their NADP(H) interaction and flavin thermodynamics, the differences were minor and unlikely to explain their distinct electron transfer activities. In contrast, calmodulin (CaM)-free eNOSr favored the FMN-shielded (electron-accepting) conformation over the FMN-deshielded (electron-donating) conformation to a much greater extent than did CaM-free nNOSr when the bound FMN cofactor was poised in each of its three possible oxidation states. NADPH binding only stabilized the FMN-shielded conformation of nNOSr, whereas CaM shifted both enzymes toward the FMN-deshielded conformation. Analysis of cytochrome c reduction rates measured within the first catalytic turnover revealed that the rate of conformational change to the FMN-deshielded state differed between eNOSr and nNOSr and was rate-limiting for either CaM-free enzyme. We conclude that the set point and regulation of the FMN conformational equilibrium differ markedly in eNOSr and nNOSr and can explain the lower electron transfer activity of eNOSr.

Highlights

Nitric oxide (NO)2 is a mediator of many cell functions [1]
When NADPH binds to enzymes like cytochrome P450 reductase (CPR) and nNOSr, a stacking interaction must occur between the nicotinamide ring and the FAD isoalloxazine ring in order for hydride transfer to be possible [22, 24, 41, 46, 47]
We found that eNOSr maintains a greater degree of FMN shielding than nNOSr, largely independent of the FMN redox state or whether

Summary

EXPERIMENTAL PROCEDURES

General Methods and Materials—All reagents and materials were obtained from Sigma, Amersham Biosciences, or other sources as reported previously [24]. A correction factor was generated from a linear regression fit of the FNR subdomain fluorescence intensities versus its concentration to obtain only the FMN subdomain fluorescence intensity in the eNOSr and nNOSr samples. This correction factor was subtracted from the total flavin fluorescence intensity of each protein. Reaction of 1-Electron Reduced NOSr Proteins with Excess Cytochrome c—1-Electron reduced eNOSr (20 ␮M) or nNOSr (5 ␮M) was obtained by adding a slight molar excess of NADPH in a cuvette and allowed to air-oxidize until the FMN semiquinone (FMNsq) was stable for 1 h. The delay time was determined from the residual and defined as the time point where there is at least 5–10% deviation from the linear fit

RESULTS

General Conclusions Regarding

DISCUSSION