A connecting hinge represses the activity of endothelial nitric oxide synthase

Abstract

In mammals, endothelial nitric oxide synthase (eNOS) has the weakest activity, being one-tenth and one-sixth as active as the inducible NOS (iNOS) and the neuronal NOS (nNOS), respectively. The basis for this weak activity is unclear. We hypothesized that a hinge element that connects the FMN module in the reductase domain but is shorter and of unique composition in eNOS may be involved. To test this hypothesis, we generated an eNOS chimera that contained the nNOS hinge and two mutants that either eliminated (P728IeNOS) or incorporated (I958PnNOS) a proline residue unique to the eNOS hinge. Incorporating the nNOS hinge into eNOS increased NO synthesis activity 4-fold, to an activity two-thirds that of nNOS. It also decreased uncoupled NADPH oxidation, increased the apparent K mO2 for NO synthesis, and caused a faster heme reduction. Eliminating the hinge proline had similar, but lesser, effects. Our findings reveal that the hinge is an important regulator and show that differences in its composition restrict the activity of eNOS relative to other NOS enzymes.electron flux ͉ heme reduction ͉ kox N itric oxide (NO) is a widespread signal molecule in biology (1, 2). Three nitric oxide synthases (NOSs) generate NO in mammals [inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS)]. All three are comprised of an Nterminal heme-containing oxygenase domain, an intervening calmodulin (CaM) binding sequence, and a C-terminal reductase domain that contains FMN and FAD (3). The three NOS enzymes have different gene expression patterns, protein interactions, posttranslational modifications, and catalytic behaviors that enable specific roles in biology (4-8). Of note, the NO synthesis activity of eNOS is one-10th that of iNOS and one-sixth that of nNOS. This activity is associated with a slower heme reduction in eNOS (9, 10). Which protein features enable these differences, and why they evolved, are still unclear.Studies of eNOS and nNOS chimeras established that their NO synthesis activities and heme reduction rates are primarily a function of the reductase domain (9, 11). For example, a chimera comprised of an eNOS oxygenase domain fused to an nNOS reductase domain had an NO synthesis activity and heme reduction rate that were similar to those of wild-type nNOS. This result implies that protein structural elements within the reductase domain are largely responsible for the different catalytic behaviors of eNOS and nNOS. While addressing this issue, we became interested in two hinge elements that connect the FMN subdomain of NOS to the rest of the enzyme (Fig. 1A). During catalysis, these hinge elements position the FMN subdomain to receive electrons from the NADPH-FAD module, and then may guide its interactions with the NOS oxygenase domain for electron transfer to the heme (12-17). In this way, the hinge elements could determine the rate of heme reduction and NO synthesis activity.The hinge that connects the FMN subdomain to the rest of the nNOS reductase domain is visible in the reductase crystal structure...

Phosphorylation of Endothelial Nitric-oxide Synthase Regulates Superoxide Generation from the Enzyme

“…from the heme is the transfer of electrons from the reductase domain to the oxygenase domain (47,48), which in the global kinetic model of NOS activity proposed by Stuehr et al (49) is the same rate-limiting step for NO production. Thus, it would be expected that any modification that alters eNOS NO production will similarly alter the activity of the uncoupled enzyme.…”

“…Along with the prior work using S1177D and ⌬27, our data support this hypothesis. However, although Ser-1177 phosphorylation enhances the eNOS-CaM interaction, this phosphorylation event also would lead to an increase in the absolute rate of electron transfer from the reductase domain to the heme with increased flavin reduction rate, a hallmark of AR inhibition release (47).…”

Regulation of Interdomain Interactions by Calmodulin in Inducible Nitric-oxide Synthase

“…In fact, the 42 residue-long eNOS C-terminal tail most likely contributes to eNOS having the weakest activity among three NOSs. One other factor contributing to the slow activity of eNOS has been shown to be the linker between the two flavin domains (61).…”

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

“…tase activity than nNOS (15,16). The basis for these differences is unclear, but it appears to involve slower electron transfer by eNOSr.…”

“…Previous studies have investigated structural elements that appear to regulate NOS activity. These include an autoinhibitory element in the FMN binding subdomain (a 40 -50-amino acid loop) (17)(18)(19), a connecting hinge domain between the FMN and FAD binding subdomains (23-25-amino acid residues) (15,20), and the C-terminal tail (21-42-amino acid extension) (16,21,22). It has also been shown that an FAD stacking residue (Phe-1160 in bovine eNOS and Phe-1395 in rat nNOS) and site-specific phosphorylation (Ser-1179 in bovine eNOS and Ser-1412 in rat nNOS) are involved in the catalytic regulation of NOS enzymes (21,(23)(24)(25).…”