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

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

doi:10.1074/jbc.m802914200

Cited by 46 publications

(166 citation statements)

References 65 publications

(115 reference statements)

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“…The mean values can also fluctuate depending on the dwell times of individual states over the course of the collection time (i.e., shorter-lived conformations may appear more times during the collection period). Nevertheless, our study provides, to our knowledge, the first dynamics-based, single molecule demonstration that under physiologic conditions, CaM binding shifts the conformational distribution of nNOSr toward more open states, consistent with results from ensemble-based studies (26,27,34,37), and we additionally show that CaM narrows the conformational population distribution of nNOSr.…”

Section: Resultssupporting

confidence: 69%

“…two to four discreet conformational populations in NOS enzymes and in related flavoproteins, and in some cases, have also estimated the distances between the bound FAD and FMN cofactors in the different species (26,36,37,39,40), and furthermore, have confirmed that CaM shifts the NOS population distribution toward more open conformations (34,36,45). Although valuable, such ensemble-averaged results about conformational states cannot explain how electrons transfer through these enzymes, or how CaM increases the electron flux in NOS, because answering these questions requires a coordinate understanding of the dynamics of the conformational fluctuations.…”

Section: Significancementioning

confidence: 71%

“…In this model, the FMN domain is suggested to be highly dynamic and flexible due to a connecting hinge that allows it to alternate between its electron-accepting (FAD→FMN) or closed conformation and electron-donating (FMN→heme) or open conformation ( Fig. 1 A and B) (28,(30)(31)(32)(33)(34)(35)(36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25,28,32,34,35,37).…”

mentioning

confidence: 99%

“…1 A and B) (28,(30)(31)(32)(33)(34)(35)(36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25,28,32,34,35,37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38)(39)(40)(41)(42).…”

mentioning

confidence: 99%

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Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Haque

Stuehr

et al. 2015

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

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Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.A lthough proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein-protein interactions (2-4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5-11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein's structural dynamics link to its functional mechanisms (12-21).A case in point is the nitric oxide synthase (NOS) enzymes (22-24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached "oxygenase" domain, which then enables NO synthesis to begin (22,(25)(26)(27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly...

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Section: Resultssupporting

confidence: 69%

Section: Significancementioning

confidence: 71%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Haque

Stuehr

et al. 2015

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

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“…In this mechanism, CaM binding releases the FMN subdomain from its interaction with the FAD/NADPH subdomain, allowing the FMN subdomain to transfer electrons to the oxidase domain (or alternative acceptors such as cytochrome c; Fig. 1B) (34)(35)(36)(37)(38)(39). Indeed, in CaM-bound nNOS, many 2D class averages were observed whereby the FMN subdomain is visualized as a blurred cloud of density (Fig.…”

Section: Discussionmentioning

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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Gaseous Mediators in Temperature Regulation

Branco

Soriano

Steiner

2014

Comprehensive Physiology

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Deep body temperature (Tb) is kept relatively constant despite a wide range of ambient temperature variation. Nevertheless, in particular situations it is beneficial to decrease or to increase Tb in a regulated manner. Under hypoxia for instance a regulated drop in Tb (anapyrexia) is key to reduce oxygen demand of tissues when oxygen availability is diminished, leading to an increased survival rate in a number of species when experiencing low levels of inspired oxygen. On the other hand, a regulated rise in Tb (fever) assists the healing process. These regulated changes in Tb are mediated by the brain, where afferent signals converge and the most important regions for the control of Tb are found. The brain (particularly some hypothalamic structures located in the preoptic area) modulates efferent activities that cause changes in heat production (modulating brown adipose tissue activity and perfusion, for instance) and heat loss (modulating tail skin vasculature blood flow, for instance). This review highlights key advances about the role of the gaseous neuromodulators nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) in thermoregulation, acting both on the brain and the periphery.

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

Cited by 46 publications

References 65 publications

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Molecular architecture of mammalian nitric oxide synthases

Gaseous Mediators in Temperature Regulation

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