Pulsed EPR Determination of the Distance between Heme Iron and FMN Centers in a Human Inducible Nitric Oxide Synthase

Astashkin, Andrei V.; Elmore, Bradley O.; Fan, Weihong; Guillemette, J. Guy; Feng, Changjian

doi:10.1021/ja104461p

Cited by 72 publications

(161 citation statements)

References 76 publications

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“…This complex has been crystallized in four different conformations showing the flexible nature of the interdomain interactions. Edge to edge distances of 13 to 18 Å between the heme iron of one subunit and the FMN center of the other subunit of the iNOS dimer were recently determined using pulse EPR experiments [63]. These distances are in excellent agreement with IET kinetic studies [64,65], low temperature MCD data [66], and support the tethered shuttle model.…”

Section: Biophysical Studies To Characterize Cam Bound To the Nos Isosupporting

confidence: 59%

Mapping the Binding and Calmodulin-Dependent Activation of Nitric Oxide Synthase Isozymes

Spratt¹,

Duangkham²,

Taiakina³

et al. 2011

TONOJ

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Calmodulin (CaM) is a ubiquitous cytosolic Ca 2+ -binding protein that can bind and activate numerous intracellular target proteins including the nitric oxide synthase (NOS) enzymes. There are three different isoforms of NOS found in mammals -neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) -with all three isozymes capable of producing the free radical, nitric oxide (•NO). At elevated cellular Ca 2+ concentrations, CaM is able to bind and activate nNOS and eNOS. In contrast, the iNOS isozyme is transcriptionally regulated and binds to CaM in the absence of Ca 2+ . To further investigate the differences in the association of CaM to the Ca 2+ -dependent and Ca 2+ -independent NOS isoforms, a variety of CaM mutants were employed. These included CaM-troponin C chimeras, CaM EF hand pair proteins, CaM mutants incapable of binding to Ca 2+ , methionine oxidized CaM mutants, phosphomimetic CaM mutants and central linker CaM mutants. By characterizing these CaM proteins using a variety of biochemical techniques, we have mapped the binding and the CaM-dependent activation of the NOS enzymes to study their effect on the different electron transfer steps within the NOS enzymes. In addition, fluorescence and Förster resonance energy transfer (FRET) were used to monitor the orientation and conformation of CaM when bound to the NOS peptides and enzymes. Combining these cumulative results, a working model describing the CaM-dependent regulation of iNOS is proposed.

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Section: Biophysical Studies To Characterize Cam Bound To the Nos Isosupporting

confidence: 59%

Mapping the Binding and Calmodulin-Dependent Activation of Nitric Oxide Synthase Isozymes

Spratt¹,

Duangkham²,

Taiakina³

et al. 2011

TONOJ

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“…Previous NOS computational modeling studies lacked the necessary experimental evidence to guide placement of calmodulin, and significant interaction between calmodulin and the heme domain had not been observed. Indeed, two studies omitted calmodulin entirely in their modeling (10,23). Whereas two other NOS modeling studies included calmodulin, neither reported direct interaction of calmodulin with the heme domain (9,11).…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies have attempted to define interactions between the heme and reductase domains using computational docking (9)(10)(11). However, selecting the catalytically relevant structure from many alternative models is difficult without direct experimental evidence delineating the protein-protein interfaces between the heme domain, FMN subdomain, and calmodulin.…”

mentioning

confidence: 99%

Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation

Smith¹,

Underbakke²,

Kulp

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

136

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Nitric oxide (NO) produced by NO synthase (NOS) participates in diverse physiological processes such as vasodilation, neurotransmission, and the innate immune response. Mammalian NOS isoforms are homodimers composed of two domains connected by an intervening calmodulin-binding region. The N-terminal oxidase domain binds heme and tetrahydrobiopterin and the arginine substrate. The C-terminal reductase domain binds FAD and FMN and the cosubstrate NADPH. Although several highresolution structures of individual NOS domains have been reported, a structure of a NOS holoenzyme has remained elusive. Determination of the higher-order domain architecture of NOS is essential to elucidate the molecular underpinnings of NO formation. In particular, the pathway of electron transfer from FMN to heme, and the mechanism through which calmodulin activates this electron transfer, are largely unknown. In this report, hydrogendeuterium exchange mass spectrometry was used to map critical NOS interaction surfaces. Direct interactions between the heme domain, the FMN subdomain, and calmodulin were observed. These interaction surfaces were confirmed by kinetic studies of site-specific interface mutants. Integration of the hydrogen-deuterium exchange mass spectrometry results with computational docking resulted in models of the NOS heme and FMN subdomain bound to calmodulin. These models suggest a pathway for electron transfer from FMN to heme and a mechanism for calmodulin activation of this critical step.iNOS | NO signaling | flavin | hemoprotein N itric oxide (NO) has several essential functions in mammalian physiology. NO produced by the neuronal and endothelial nitric oxide synthase isoforms (nNOS and eNOS, respectively) initiates diverse signaling processes including vasodilation, myocardial function, and neurotransmission (1). The eNOS and nNOS isoforms are constitutively expressed and their activity responds to intracellular calcium concentrations. The inducible NOS isoform (iNOS) is transcriptionally controlled and produces NO as a cytotoxin at sites of inflammation or infection. Aberrant NO signaling contributes to a variety of diseases including stroke, hypertension, and neurodegeneration (2).Mammalian NOS isoforms are homodimeric and composed of two principal domains: the N-terminal oxidase domain and C-terminal reductase domain, which are connected by an intervening calmodulin (CaM) binding region (Fig. 1A). The N-terminal oxidase domain contains the heme and tetrahydrobiopterin cofactors and the binding site for the substrate arginine. The reductase domain is further divided into the FMN-binding subdomain and the FAD/NADPH-binding subdomains. This array of cofactors works in concert to catalyze the conversion of arginine to the intermediate N-hydroxyarginine and, ultimately, citrulline and NO. NADPH and oxygen are consumed in the process. During catalysis, electrons are shuttled from the reductase domain of one monomer to the heme domain of the opposite monomer in the homodimer (Fig. 1B) (1, 3). Electron transfer is initiat...

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“…Structures of the nNOS reductase domain (8,16) and the iNOS FMN subdomain and CaM-binding helix bound to CaM (17) have also been reported. Previous computational docking studies (7,9,17,18) provide an emerging view of the output state in NOS holoenzymes, but these methods provide numerous models, and selecting the catalytically relevant structure or structures without direct experimental evidence is difficult. Our recent HDX-MS studies provided detailed models of the iNOS output state, but, because the FAD/NADPH subdomain was not included in the truncations used in this study, the relative positioning of the entire reductase and oxidase domains could not be determined (7).…”

Section: Significancementioning

confidence: 99%

Molecular architecture of mammalian nitric oxide synthases

Campbell

Smith

Potter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

122

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NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate immune response. Mammals possess three NOS isoformsinducible, endothelial, and neuronal NOS-that are composed of an N-terminal oxidase domain and a C-terminal reductase domain. Calmodulin (CaM) activates NO synthesis by binding to the helical region connecting these two domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length NOS. We used high-throughput single-particle EM to obtain the structures and higher-order domain organization of all three NOS holoenzymes. The structures of inducible, endothelial, and neuronal NOS with and without CaM bound are similar, consisting of a dimerized oxidase domain flanked by two separated reductase domains. NOS isoforms adopt many conformations enabled by three flexible linkers. These conformations represent snapshots of the continuous electron transfer pathway from the reductase domain to the oxidase domain, which reveal that only a single reductase domain participates in electron transfer at a time, and that CaM activates NOS by constraining rotational motions and by directly binding to the oxidase domain. Direct visualization of these large conformational changes induced during electron transfer provides significant insight into the molecular underpinnings governing NO formation.heme | flavin | electron microscopy | conformational heterogeneity N O has emerged as an integral signaling molecule in biology.NOSs are the only enzymes responsible for NO production in mammals. Disruptions in NO signaling are linked to hypertension, erectile dysfunction, neurodegeneration, stroke, and heart disease (1-3). Three NOS isoforms, which vary slightly in size and composition from 260 to 321 kDa for the NOS homodimer, are present in mammals; each is a distinct gene product differing in subcellular localization, tissue distribution, and mode of regulation. The constitutively expressed NOS isoforms are found primarily in endothelial cells [endothelial NOS (eNOS)] and neuronal cells [neuronal NOS (nNOS)], and NO produced by these isoforms initiates diverse signaling processes, including vasodilation, platelet aggregation, myocardial functions, and neurotransmission (4). The activity of the constitutive NOS isoforms is dependent on intracellular Ca 2+ concentrations. The third isoform, inducible NOS (iNOS), is transcriptionally controlled and produces cytotoxic concentrations of NO at sites of infection or inflammation. Unlike eNOS and nNOS, the activity of iNOS is independent of the intracellular Ca 2+ concentration.Mammalian NOS isoforms use a complex assembly of domains and cofactors to convert L-arginine to L-citrulline and NO, via the intermediate N-hydroxy-L-arginine. Mammalian NOS isoforms are active as homodimers and composed of two primary domains: the N-terminal oxidase domain and the C-terminal reductase domain (Fig. 1A). The re...

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Pulsed EPR Determination of the Distance between Heme Iron and FMN Centers in a Human Inducible Nitric Oxide Synthase

Cited by 72 publications

References 76 publications

Mapping the Binding and Calmodulin-Dependent Activation of Nitric Oxide Synthase Isozymes

Mapping the Binding and Calmodulin-Dependent Activation of Nitric Oxide Synthase Isozymes

Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation

Molecular architecture of mammalian nitric oxide synthases

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