Control of Electron Transfer and Catalysis in Neuronal Nitric-oxide Synthase (nNOS) by a Hinge Connecting Its FMN and FAD-NADPH Domains

Haque, Mohammad Mahfuzul; Fadlalla, Mohammed; Aulak, Kulwant S.; Ghosh, Arnab; Durra, Deborah; Stuehr, Dennis J.

doi:10.1074/jbc.m112.339697

Cited by 25 publications

(35 citation statements)

References 57 publications

(70 reference statements)

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“…Flanking hinge elements are thought to enable the FMN to shift between shielded and deshielded states to favor respective NADPH-FAD-FMN and FMN-heme electron transfer (22). Although the requirement of CaM binding is unique to NOS, flexibility of the FAD-FMN hinge has been observed in several cytochrome P450 structures and is known to be important to catalytic function (7,41).…”

Section: Discussionmentioning

confidence: 99%

“…CaM interactions increase FMNheme electron transfer, possibly by destabilizing the shielded state through bridging contacts with the FMN or interactions with the reductase connecting domain (CD) (16 -20). Electrostatic interactions via structural control elements in the reductase domain, including the autoinhibitory loop, C-terminal tail, and connecting domain, favor the shielded state and are proposed to become disrupted for precise release of the FMN domain during the catalytic cycle (21,22).…”

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confidence: 99%

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Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain

Yokom¹,

Morishima²,

Lau³

et al. 2014

Journal of Biological Chemistry

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Background: NOS enzymes are large, dimeric complexes essential in mammalian physiology. Results: EM structural analysis and three-dimensional models reveal nNOS reductase-oxygenase arrangements and a CaMdependent rotation of the FMN domain. Conclusion: Coordinated conformational changes act to reposition the FMN domain for electron transfer. Significance: This work captures structural states of the NOS holoenzyme that drive the NO synthesis cycle.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain

Yokom¹,

Morishima²,

Lau³

et al. 2014

Journal of Biological Chemistry

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“…Molecular Biology-Restriction digestions, cloning, bacterial growth, transformation, and isolation of DNA fragments were performed using standard techniques (9,28,53). The bacterial expression vector pCWori contained cDNA that coded for either bovine eNOSr (with its adjacent N-terminal CaM binding site, amino acids 445-1205) or full-length eNOS.…”

Section: Methodsmentioning

confidence: 99%

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

Haque¹,

Ray²,

Stuehr

2016

Journal of Biological Chemistry

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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...

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“…The NOSoxy domain binds a heme prosthetic group and the redox factor tetrahydrobiopterin (H 4 B) (4,5). The NOSred domain displays similarities with NADPH-cytochrome P450 reductase and transfers NADPH-derived electrons to the heme through flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) during catalysis (5)(6)(7)(8). In addition, human NOS1 has an N-terminal PDZ domain that mediates protein-protein interactions (9).…”

Section: Introductionmentioning

confidence: 99%

Occurrence, structure, and evolution of nitric oxide synthase–like proteins in the plant kingdom

et al. 2016

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Nitric oxide (NO) signaling regulates various physiological processes in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase (NOS) enzymes. Although NOS-like activities that are sensitive to mammalian NOS inhibitors have been detected in plant extracts, few bona fide plant NOS enzymes have been identified. We searched the data set produced by the 1000 Plants (1KP) international consortium for the presence of transcripts encoding NOS-like proteins in over 1000 species of land plants and algae. We also searched for genes encoding NOS-like enzymes in 24 publicly available algal genomes. We identified no typical NOS sequences in 1087 sequenced transcriptomes of land plants. In contrast, we identified NOS-like sequences in 15 of the 265 algal species analyzed. Even if the presence of NOS enzymes assembled from multipolypeptides in plants cannot be conclusively discarded, the emerging data suggest that, instead of generating NO with evolutionarily conserved NOS enzymes, land plants have evolved finely regulated nitrate assimilation and reduction processes to synthesize NO through a mechanism different than that in animals.

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Control of Electron Transfer and Catalysis in Neuronal Nitric-oxide Synthase (nNOS) by a Hinge Connecting Its FMN and FAD-NADPH Domains

Cited by 25 publications

References 57 publications

Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain

Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain

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

Occurrence, structure, and evolution of nitric oxide synthase–like proteins in the plant kingdom

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