Formation of a Pterin Radical in the Reaction of the Heme Domain of Inducible Nitric Oxide Synthase with Oxygen

Hurshman, Amy R; Krebs, Carsten; Edmondson, Dale E.; Huynh, Boi Hanh; Marletta, Michael A.

doi:10.1021/bi992026c

Cited by 221 publications

(261 citation statements)

References 46 publications

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“…Strong interaction between the spins of the pterin and the adjacent heme can also be deduced from the microwave power saturation behavior, with all pterin radicals reported thus far exhibiting remarkably high P 1/2 values (8,10,12,32). We derived fitting parameters of 1.0 milliwatts for P 1/2 and 0.77 for b, suggesting strong dipolar coupling between BH3 and high spin ferric heme (33).…”

Section: Resultsmentioning

confidence: 70%

Single-turnover of Nitric-oxide Synthase in the Presence of 4-Amino-tetrahydrobiopterin

Sørlie

Gorren

Marchal

et al. 2003

Journal of Biological Chemistry

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Tetrahydrobiopterin (BH4) is an essential cofactor of nitric-oxide synthase (NOS) that serves as a one-electron donor to the oxyferrous⅐heme complex. 4-Aminotetrahydrobiopterin (4-amino-BH4) is a potent inhibitor of NO synthesis, although it mimics all allosteric and structural effects of BH4 and exhibits comparable redox properties. We studied the reaction of reduced endothelial NOS oxygenase domain with O 2 in the presence of 4-amino-BH4 at ؊30°C by optical and electron paramagnetic resonance (EPR) spectroscopy. With Arg as the substrate, we observed a trihydropteridine radical with a corresponding heme species that was oxyferrous, with a Soret maximum at 428 nm and no EPR signal. With N G -hydroxy-L-arginine (NHA) no pterin radical appeared, whereas an axial ferrous heme⅐NO complex was formed. The corresponding optical spectra, with Soret bands at 417/423 nm, suggest that the proximal sulfur ligand is protonated. Accordingly, 4-amino-BH4 serves as a one-electron donor to Fe(II)⅐O 2 with both Arg and NHA, but the reaction cycle cannot be completed with either substrate. We propose that protonation of Fe(II)O 2 ؊ is inhibited in the presence of 4-amino-BH4. With Arg, dissociation of O 2 ؊ and binding of O 2 yields Fe(II)⅐O 2 and a pteridine radical; with NHA, reaction of the substrate with heme-bound O 2 ؊ eventually yields Fe(II)⅐NO and reduced 4-amino-BH4. These results suggest that BH4 donates a proton to Fe(II)⅐O 2 ؊ during catalysis and that inhibition by 4-amino-BH4 may be due to its inability to support this essential protonation step.

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Section: Resultsmentioning

confidence: 70%

Single-turnover of Nitric-oxide Synthase in the Presence of 4-Amino-tetrahydrobiopterin

Sørlie

Gorren

Marchal

et al. 2003

Journal of Biological Chemistry

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“…In mammalian NOSs, H 4 B is implicated as an electron donor (32,39) and provides an electron to the heme FeIIO 2 intermediate for Arg hydroxylation in a single turnover reaction (31). In this circumstance electron transfer from H 4 B speeds disappearance of the FeIIO 2 intermediate (22).…”

Section: Discussionmentioning

confidence: 99%

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

Adak

Bilwes

Panda

et al. 2001

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

122

139

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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...

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“…The EPR spectrum in the absence of NADPH (Fig. 4) is indicative of a high spin Fe(III) heme species (14). The absence of a signal from the 2Fe2S cluster indicates that it is in a S ϭ 0, [Fe 2 S 2 ] 2ϩ state.…”

Section: Investigation Of Scnos Reduction With Nadph By Epr and Uv-vismentioning

confidence: 98%

“…The reductase domain transfers electrons from NADPH (nicotinamide adenine dinucleotide phosphate) via FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) to the P450-type heme in the oxygenase domain (13). NOS contains an additional reduced pterin cofactor in the oxygenase domain [H 4 B, (6R)-tetrahydro-l-biopterin], which is required for NO formation and is involved in electron transfer processes during catalysis at the heme (14)(15)(16).…”

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confidence: 99%

NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum

Agapie

Suseno²,

Woodward³

et al. 2009

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

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The role of nitric oxide (NO) in the host response to infection and in cellular signaling is well established. Enzymatic synthesis of NO is catalyzed by the nitric oxide synthases (NOSs), which convert Arg into NO and citrulline using co-substrates O2 and NADPH. Mammalian NOS contains a flavin reductase domain (FAD and FMN) and a catalytic heme oxygenase domain (P450-type heme and tetrahydrobiopterin). Bacterial NOSs, while much less studied, were previously identified as only containing the heme oxygenase domain of the more complex mammalian NOSs. We report here on the characterization of a NOS from Sorangium cellulosum (both fulllength, scNOS, and oxygenase domain, scNOSox). scNOS contains a catalytic, oxygenase domain similar to those found in the mammalian NOS and in other bacteria. Unlike the other bacterial NOSs reported to date, however, this protein contains a fused reductase domain. The scNOS reductase domain is unique for the entire NOS family because it utilizes a 2Fe2S cluster for electron transfer. scNOS catalytically produces NO and citrulline in the presence of either tetrahydrobiopterin or tetrahydrofolate. These results establish a bacterial electron transfer pathway used for biological NO synthesis as well as a unique flexibility in using different tetrahydropterin cofactors for this reaction.heme protein ͉ iron-sulfur cluster ͉ reductase ͉ tetrahydrobiopterin ͉ tetrahydrofolate N itric oxide (NO) is recognized as an important signaling molecule as well as a cytotoxin (1-3). A number of diseases are intimately tied to improper function of NO in humans (1-3). Given the importance of NO to human health, a wealth of studies has been performed to address questions regarding NO synthesis and regulation (4-9). Isolation and structural characterization of the proteins responsible for NO synthesis have led to important developments in understanding the molecular processes of NO formation (10-12). Three isoforms of nitric oxide synthase (NOS), iNOS, eNOS, and nNOS, have been characterized in mammals. These enzymes contain an oxygenase domain where the catalysis takes place and a reductase domain that is involved in electron transfer (4, 9). NOS is functional only as a homodimer, and requires the binding of a Ca 2ϩ -calmodulin (CaM) complex for electron transfer between the reductase and oxygenase domains. The reductase domain transfers electrons from NADPH (nicotinamide adenine dinucleotide phosphate) via FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) to the P450-type heme in the oxygenase domain (13). NOS contains an additional reduced pterin cofactor in the oxygenase domain [H 4 B, (6R)-tetrahydro-l-biopterin], which is required for NO formation and is involved in electron transfer processes during catalysis at the heme (14-16).NOS catalyzes the conversion of Arg into NO and citrulline using O 2 and NADPH involving two catalytic steps. In the first reaction, Arg is oxidized to N G -hydroxy-arginine (NHA). Nitric oxide is generated in the second half of the cycle, with the conversion...

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Formation of a Pterin Radical in the Reaction of the Heme Domain of Inducible Nitric Oxide Synthase with Oxygen

Cited by 221 publications

References 46 publications

Single-turnover of Nitric-oxide Synthase in the Presence of 4-Amino-tetrahydrobiopterin

Single-turnover of Nitric-oxide Synthase in the Presence of 4-Amino-tetrahydrobiopterin

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

NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum

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