Phosphorylation Controls Endothelial Nitric-oxide Synthase by Regulating Its Conformational Dynamics

Abstract

The activity of endothelial NO synthase (eNOS) is triggered by calmodulin (CaM) binding and is often further regulated by phosphorylation at several positions in the enzyme. Phosphorylation at Ser 1179 occurs in response to diverse physiologic stimuli and increases the NO synthesis and cytochrome c reductase activities of eNOS, thereby enhancing its participation in biological signal cascades. Despite its importance, the mechanism by which Ser 1179 phosphorylation increases eNOS activity is not understood. To address this, we used stopped-flow spectroscopy and computer modeling approaches to determine how the phosphomimetic mutation (S1179D) may impact electron flux through eNOS and the conformational behaviors of its reductase domain, both in the absence and presence of bound CaM. We found that S1179D substitution in CaM-free eNOS had multiple effects; it increased the rate of flavin reduction, altered the conformational equilibrium of the reductase domain, and increased the rate of its conformational transitions. We found these changes were equivalent in degree to those caused by CaM binding to wild-type eNOS, and the S1179D substitution together with CaM binding caused even greater changes in these parameters. The modeling indicated that the changes caused by the S1179D substitution, despite being restricted to the reductase domain, are sufficient to explain the stimulation of both the cytochrome c reductase and NO synthase activities of eNOS. This helps clarify how Ser 1179 phosphorylation regulates eNOS and provides a foundation to compare its regulation by other phosphorylation events.Nitric oxide (NO) synthase enzymes (EC 1.14.13.39) are homodimers with subunits composed of an N-terminal oxygenase domain (NOSoxy) 4 that is linked to a C-terminal reductase domain (NOSr) by an intervening calmodulin (CaM) binding sequence (1-4). Many of the structural and catalytic features of NOSr are shared among a family of eukaryotic dual-flavin enzymes whose members include cytochrome P450 reductase, methionine synthase reductase, and novel reductase-1 (5-7). These enzymes are all composed of an FMN-binding domain that is attached to a FAD/NADPH-binding domain (ferredoxin NADP ϩ -reductase (FNR)) by a flexible hinge of various lengths (8 -10). Their electron transfer (ET) reactions all involve a hydride transfer from NADPH to the FAD cofactor bound within the FNR domain, which then passes electrons sequentially to the FMN domain. Once the FMN domain receives two electrons, its FMN hydroquinone (FMNhq) can transfer an electron to a heme group that is located either within the enzyme itself (i.e. NOS), in a partner hemeprotein acceptor (i.e. cytochrome P450 reductase), or in a non-native hemeprotein acceptor such as cytochrome c (6,(11)(12)(13)(14). Importantly, ET through diflavin enzymes relies on the transient interactions and motions of the FMN domain, which must move between an FNR-bound, conformationally closed "input" state and an unbound, conformationally open "output" state (15-17). We and others have proposed...

Restricting the conformational freedom of the neuronal nitric-oxide synthase flavoprotein domain reveals impact on electron transfer and catalysis

“…This implies that a change in FMN domain conformational behavior is needed for CaM to have its kinetic effect on flavin reduction. A recent study showed that CaM speeds the conformational dynamics of nNOSr (16), and this effect, in light of our current results, can explain how CaM could speed flavin reduction in nNOSr but fail to do so in the disulfide-linked CLSS. The disulfide cross-linked CLSS greatly, but not completely, lost its ability to catalyze ET to cytochrome c. We suspect that its residual activity is due to catalysis by the disulfide crosslinked CLSS itself, rather than any contamination by noncross-linked CLSS.…”

“…As catalysis continues, the rates of conformational cycling and interflavin ET all contribute to determining the flux of electrons going to cytochrome c during steady-state catalysis. Using this approach, we have studied how conformational behavior controls electron flux through various NOS proteins (10,16,35) and through related dual-flavin reductase enzymes like CPR and methionine synthase reductase. Fig.…”

“…Thus, during catalysis the FMN domain is thought to move so that the enzyme can cycle between closed and more open conformational states to support ET to its protein partners (6,12). Indeed, a variety of spectroscopic and biophysical studies has shown that open conformational states exist in equilibrium with the closed state in NOS (13)(14)(15)(16)(17)(18)(19) and in CPR (20 -24), and recently the crystal structures of three open conformations have been reported for CPR mutants or chimeras (25,26).…”

Autoinflammation in the pathogenesis of gout and hyperuricemia

“…Среди механизмов артериальной гипертонии у больных с гиперурикемией в первую очередь обсуждают блокаду продукции оксида азота [14][15][16][17][18][19][20][21][22][23][24], в результате чего снижается его системное вазодилатирующее действие, уменьшаются контроль пролиферативной активности гладких миоцитов стенок артерий и антитромбогенный эффект. Возникающее под влиянием этого повышение общего периферического сопротивления сосудов является мощным фактором активации ренин-ангиотензиновой системы с развитием артериальной гипертонии.…”

“…Возникающее под влиянием этого повышение общего периферического сопротивления сосудов является мощным фактором активации ренин-ангиотензиновой системы с развитием артериальной гипертонии. Блокаду синтеза оксида азота у больных с гиперурикемией связывают с гиперпродукцией свободных перекисных радикалов, что, в свою очередь, обусловлено персистирующей субклинической активацией аутовоспалительных механизмов [14][15][16][17][18][19][20][21][22][23][24]. H. Zheng и соавт.…”