Role of Reductase Domain Cluster 1 Acidic Residues in Neuronal Nitric-oxide Synthase

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We cloned, expressed, and characterized a hemeprotein from Deinococcus radiodurans (D. radiodurans NO synthase, deiNOS) whose sequence is 34% identical to the oxygenase domain of mammalian NO synthases (NOSoxys). deiNOS was dimeric, bound substrate Arg and cofactor tetrahydrobiopterin, and had a normal heme environment, despite its missing N-terminal structures that in NOSoxy bind Zn 2؉ and tetrahydrobiopterin and help form an active dimer. The deiNOS heme accepted electrons from a mammalian NOS reductase and generated NO at rates that met or exceeded NOSoxy. Activity required bound tetrahydrobiopterin or tetrahydrofolate and was linked to formation and disappearance of a typical heme-dioxy catalytic intermediate. Thus, bacterial NOS-like proteins are surprisingly similar to mammalian NOSs and broaden our perspective of NO biochemistry and function.G enes coding for NO synthases (NOSs, EC 4.14.23) are present throughout the plant and animal kingdom. NOS activities are present in plants and lower eukaryotes (1-5). Their primary structures and activities are strikingly similar to the mammalian NOSs, suggesting that NO has been important throughout evolution. All eukaryotic NOSs catalyze the NADPH-and O 2 -dependent oxidation of L-arginine (Arg) to citrulline and NO, with N-hydroxy-L-arginine (NOHA) formed as an enzyme-bound intermediate (6). Structurally, all animal NOSs are bi-domain proteins containing an N-terminal oxygenase domain (NOSoxy) that binds protoporphyrin IX (heme), 6R-tetrahydrobiopterin (H 4 B), and Arg and is linked to a C-terminal f lavoprotein domain (NOS reductase domain, NOSred) by a central calmodulin (CaM) binding sequence (7,8). NOSred bears strong sequence and functional similarity to NADPH-cytochrome P450 reductase and related electron transfer flavoproteins (9, 10), and function to transfer NADPHderived electrons to the ferric heme for O 2 activation during NO synthesis. In contrast, NOSoxy and cytochromes P450 have completely different primary, secondary, and tertiary structures, even though both enzyme families use a thiolate-ligated heme for O 2 activation (11, 12). Moreover, unlike P450s, NOSoxy must dimerize to become active (13,14). Dimerization produces functional binding sites for Arg and H 4 B and sequesters the heme catalytic center from solvent. These distinguishing features imply that NOSs evolved separately from other heme-thiolate enzymes and can so provide unique perspectives on their structure-function relationships.Although oxidative (nitrification) or reductive (denitrification) pathways for prokaryote NO biosynthesis are well established (15), there have been few reports on NOS-like proteins in bacteria (16,17). To date no prokaryotic NOS proteins have been completely sequenced, purified, or cloned. However, some have been shown to have nitrite-forming activity that depended on Arg, NADPH, and H 4 B. More recent genome sequencing of Bacillus subtilis, Deinococcus radiodurans, and other bacteria (18)(19)(20) confirm that a subset contain ORFs that code for proteins homo...

Section: Methodsmentioning

confidence: 99%

Cloning, expression, and characterization of a nitric oxide synthase protein from Deinococcus radiodurans

Adak

Bilwes

Panda

et al. 2001

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122

139

“…Restriction digestions, cloning, bacterial growth, transformation, and isolation of DNA fragments were performed by using standard procedures. Rat nNOS DNA was inserted into the 5Ј NdeI and 3Ј XbaI site into the pCWori vector (32)(33)(34). The F1395Y and F1395S mutation site in the nNOS cDNA was constructed by subcloning a PCR-generated fragment from pCWori͞nNOS with a 5Ј oligonucleotide containing a newly engineered mutational site.…”

Section: Methodsmentioning

confidence: 99%

“…The nNOS cDNA fragment coding from the KpnI unique restriction site at position 4514 to the XbaI restriction site at position 4785 was amplified by using to their N termini to aid purification. They were overexpressed in E. coli strain BL21(DE3) and purified by sequential chromatography on Ni 2ϩ -NTA and 2Ј,5Ј-ADP-Sepharose as described (34).…”

Section: Methodsmentioning

confidence: 99%

A conserved flavin-shielding residue regulates NO synthase electron transfer and nicotinamide coenzyme specificity

Adak

Sharma

Meade

et al. 2002

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Nitric oxide synthases (NOSs) are flavoheme enzymes that contain a ferredoxin:NADP ؉ -reductase (FNR) module for binding NADPH and FAD and are unusual because their electron transfer reactions are controlled by the Ca 2؉ -binding protein calmodulin. A conserved aromatic residue in the FNR module of NOS shields the isoalloxazine ring of FAD and is known to regulate NADPH binding affinity and specificity in related flavoproteins. We mutated Phe-1395 (F1395) in neuronal NOS to Tyr and Ser and tested their effects on nucleotide coenzyme specificity, catalytic activities, and electron transfer in the absence or presence of calmodulin. We found that the aromatic side chain of F1395 controls binding specificity with respect to NADH but does not greatly affect affinity for NADPH. Measures of flavin and heme reduction kinetics, ferricyanide and cytochrome c reduction, and NO synthesis established that the aromatic side chain of F1395 is required to repress electron transfer into and out of the flavins of neuronal NOS in the calmodulin-free state, and is also required for calmodulin to fully relieve this repression. We speculate that the phenyl side chain of F1395 is part of a conformational trigger mechanism that negatively or positively controls NOS electron transfer depending on the presence of calmodulin. Such use of the conserved aromatic residue broadens our understanding of flavoprotein structure and regulation.N itric oxide (NO) is generated by NO synthases (NOSs) and modulates physiology and pathology in mammals (1, 2). The NOS polypeptide consists of an N-terminal oxygenase domain that contains heme, 6R-tetrahydrobiopterin (H 4 B), and an Larginine (Arg) binding site, and a C-terminal reductase domain that contains FMN, FAD, and an NADPH binding site (3-5). An Ϸ20-aa calmodulin (CaM) binding site is located between the reductase and oxygenase domains (3-5). During NO synthesis, electrons from NADPH transfer in a linear sequence to FAD, FMN, and then to the heme, which binds and activates dioxygen (3-5). Although NOS oxygenase domains have unusual structure (6-8), NOS reductase domains are structurally similar to other NADPH-requiring flavoproteins like cytochrome P450 reductase (CYPR), sulfite reductase flavoprotein, methionine synthase reductase, and cytochrome P450BM3 (9-11). These flavoproteins are all comprised of a flavodoxin module that contains FMN and a ferredoxin:NADP ϩ reductase (FNR) module that contains FAD and the NADPH binding site (12).NOS catalysis is controlled by varied mechanisms (3-5, 13). Surprisingly, its electron transfer events are regulated by Ca 2ϩ -dependent CaM binding (14). Although this is unusual for a redox enzyme, it may be representative of a growing class of Ca 2ϩ -activated flavoproteins that include NADPH oxidases (15) and a flavoprotein of Desulfovibrio gigas (16). In NOS, CaM binding relieves a kinetic repression on electron transfer both into and out of its reductase domain (17, 18). We are only beginning to understand the basis for the repression or how it is relieved by ...

“…To transition into the output state, a large conformational change occurs that allows the FMN subdomain to donate an electron to the oxidase domain. Although progress has been made elucidating the interaction surfaces present in the input and output states (7)(8)(9)(10)(11)(12), the trajectory of this conformational change between the input and output is only beginning to be explored (13). Further understanding of this conformational change is particularly important as electron transfer is rate-limiting during NOS catalysis (6).…”

mentioning

confidence: 99%

Molecular architecture of mammalian nitric oxide synthases

Campbell

Smith

Potter

et al. 2014

113

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...