Endothelial nitric-oxide synthase (eNOS) plays important roles in vascular physiology and homeostasis. Whether eNOS catalyzes nitric oxide biosynthesis or the synthesis of reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite is dictated by the bioavailability of tetrahydrobiopterin (BH 4 ) and L-arginine during eNOS catalysis. The effect of BH 4 and Larginine on oxygen-induced radical intermediates has been investigated by single turnover rapid-freeze quench and EPR spectroscopy using the isolated eNOS oxygenase domain (eNOS ox ). Three distinct radical intermediates corresponding to >50% of the heme were observed during the reaction between ferrous eNOS ox and oxygen. Nitric-oxide synthase (NOS), 1 which catalyzes nitric oxide (NO) biosynthesis, is a complicated cytochrome P450-like enzyme. Three substrates (L-arginine, NADPH, and oxygen) and four products (NO, L-citrulline, NADPϩ, and water) are involved in the 5-electron oxidation of the guanidino nitrogen of L-arginine to yield NO. Four cofactors, FMN and FAD in the reductase domain and heme and BH 4 in the oxygenase domain, participate in catalysis (1, 2). The reductase domain mainly functions as the source of reducing equivalents generated after NADPH binding, and the heme center is the site of the key chemical events. A tight coupling between the reductase and oxygenase domains maximizes NO formation rather than producing side products such as superoxide, hydrogen peroxide, and even peroxynitrite (3-6). When NOS behaves as a superoxide synthase or peroxynitrite synthase, it may cause endothelial dysfunction, arteriosclerosis, and even vascular injury and septic shock (7-10). The two major controlling factors in achieving an optimal redox coupling are the timely supply of L-arginine and the integration of BH 4 into the oxygenase domain. Superoxide radical formation in three different NOS isoforms has been studied extensively in the presence or absence of either L-arginine or BH 4 mainly by spin-trapping and EPR spectroscopy (3,4,(11)(12)(13). It was found that autoxidation of flavin in the reductase domain dominates superoxide formation in iNOS (11), whereas breakdown of the Fe(II)O 2 (or Fe(III)O 2 Ϫ ) heme intermediate is the main source of superoxide in nNOS and eNOS (3,4,12). Furthermore, superoxide formation in nNOS is almost completely inhibited by L-arginine, whereas in eNOS only BH 4 appears to suppress superoxide formation fully (3,4,12). It is critical to understand these isozyme-specific regulatory processes of eNOS to develop strategies to intervene in NOS-derived vascular dysfunction in an isozyme-specific manner.Several experimental barriers hamper elucidation of the underlying mechanisms regarding the differential regulation of superoxide formation by L-arginine and/or BH 4 . First, most previous studies were carried out under steady-state conditions, which involve many complicated chemical steps from both the reductase and the oxygenase domains, and it is difficult to locate the key steps that define the kinetics...

Prostacyclin is a potent mediator of vasodilation and anti-platelet aggregation. It is synthesized from prostaglandin H(2) by prostacyclin synthase (PGIS), a member of Family 8 in the cytochrome P450 superfamily. Unlike most P450s, which require exogenous reducing equivalents and an oxygen molecule for mono-oxygenation, PGIS catalyzes an isomerization with an initial step of endoperoxide bond cleavage of prostaglandin H(2) (PGH(2)). The low abundance of PGIS in natural tissues necessitates heterologous expression for studies of structure/function relationships and reaction mechanism. We report here a high-yield prokaryotic system for expression of enzymatically active human PGIS. The PGIS cDNA is modified by replacing the hydrophobic amino-terminal sequence with the more hydrophilic amino-terminal sequence from P450 2C5 and by adding a four-histidine tag at the carboxyl terminus. The resulting recombinant PGIS associates with host cell membranes and was purified to electrophoretic homogeneity by nickel affinity, hydroxyapatite and CM Sepharose column chromatography. The recombinant PGIS, with a heme:protein ratio of 0.9:1, catalyzes prostacyclin formation at a K(m) of 13.3 muM PGH(2) and a V(max) of 980 per min. The dithionite-reduced PGIS binds CO with an on-rate of 5.6 x 10(5) M(-1) s(-1) and an off-rate of 15 s(-1). The ferrous-CO complex of PGIS is very short-lived and decays at a rate of 0.7 s(-1). Spectral binding assays showed that imidazole binds weakly to PGIS (K(d) approximately 0.5 mM,) but clotrimazole, a bulky and rigid imidazole derivative, binds strongly (K(d) approximately 1 microM). The transient nature of the CO complex and the weak imidazole binding seem to support an earlier proposal that PGIS active site has a limited space, but the tight binding of clotrimazole argues against this view. It appears that the heme distal pocket of PGIS is fairly adaptable to ligands of various structures. UV-visible absorption, magnetic circular dichroism and electron paramagnetic resonance spectra indicate that PGIS has a typical low-spin heme with a hydrophobic active site. PGIS catalyzes homolytic scission of the peroxide bond of a test substrate, 10-hydroperoxyoctadeca-8,12-dienoic acid, accompanied by formation of a heme intermediate with a Compound II-like optical spectrum.

Characterization of the redox properties of endothelial nitric-oxide synthase (eNOS) is fundamental to understanding the complicated reaction mechanism of this important enzyme participating in cardiovascular function. Yeast overexpression of both the oxygenase and reductase domains of human eNOS, i.e. eNOS ox and eNOS red , has been established to accomplish this goal. UV-visible and electron paramagnetic resonance (EPR) spectral characterization for the resting eNOS ox and its complexes with various ligands indicated a standard NOS heme structure as a thiolate hemeprotein. Two low spin imidazole heme complexes but not the isolated eNOS ox were resolved by EPR indicating slight difference in heme geometry of the dimeric eNOS ox domain. Stoichiometric titration of eNOS ox demonstrated that the heme has a capacity for a reducing equivalent of 1-1.5. Additional 1.5-2.5 reducing equivalents were consumed before heme reduction occurred indicating the presence of other unknown high potential redox centers. There is no indication for additional metal centers that could explain this extra electron capacity of eNOS ox . Ferrous eNOS ox , in the presence of L-arginine, is fully functional in forming the tetrahydrobiopterin radical upon mixing with oxygen as demonstrated by rapidfreeze EPR measurements. Calmodulin binds eNOS red at 1:1 stoichiometry and high affinity. Stoichiometric titration and computer simulation enabled the determination for three redox potential separations between the four half-reactions of FMN and FAD. The extinction coefficient could also be resolved for each flavin for its semiquinone, oxidized, and reduced forms at multiple wavelengths. This first redox characterization on both eNOS domains by stoichiometric titration and the generation of a high quality EPR spectrum for the BH 4 radical intermediate illustrated the usefulness of these tools in future detailed investigations into the reaction mechanism of eNOS. Nitric-oxide synthase (NOS)1 is an uncommon self-sufficient P450-like enzyme catalyzing nitric oxide (NO) biosynthesis from L-arginine (1-4). There are three mammalian NOS isozymes: the constitutive neuronal NOS (nNOS) and endothelial NOS (eNOS) require calmodulin for enzyme activity, whereas the inducible NOS (iNOS) contains tightly bound calmodulin (1-4). All three isozymes have a common bi-domain structure with the reductase domain containing FAD, FMN, and NADPH binding sites, and the oxygenase domain harboring the heme center and binding sites for L-arginine and tetrahydrobiopterin (BH 4 ) (1-4). The main function of the reductase domain is to provide reducing equivalents to the heme center in the oxygenase domain where the key chemistry of L-arginine conversion occurs. Three substrates and four products are involved in NOS catalysis. The overall reaction is a complicated five-electron oxidation of the key guanidine nitrogen plus three additional electrons from NADPH to reduce two molecules of oxygen to water and form the L-citrulline and nitric oxide. Several x-ray crystallographic s...

Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) are atypical cytochrome P450s. They do not require NADPH or dioxygen for isomerization of prostaglandin H 2 (PGH 2 ) to produce prostacyclin (PGI 2 ) and thromboxane A 2 (TXA 2 ). PGI 2 and TXA 2 have opposing actions on platelet aggregation and blood vessel tone. In this report, we use a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), to explore the active site characteristics of PGIS and TXAS. The two enzymes transformed 15-HPETE not only into 8,, like many microsomal P450s, but also to 15-ketoeicosatetraenoic acid (15-KETE) and 15-hydroxyeicosatetraenoic acid (15-HETE). 13-OH-14,15-EET and 15-KETE result from homolytic cleavage of the O-O bond, whereas 15-HETE results from heterolytic cleavage, a common peroxidase pathway. About 80% of 15-HPETE was homolytically cleaved by PGIS and 60% was homolytically cleaved by TXAS. The V max of homolytic cleavage is 3.5-fold faster than heterolytic cleavage for PGIS-catalyzed reactions (1100 min −1 vs. 320 min −1 ) and 1.4-fold faster for TXAS (170 min −1 vs. 120 min −1 ). Similar K M values for homolytic and heterolytic cleavages were found for PGIS (∼60 μM 15-HPETE) and TXAS (∼80 μM 15-HPETE), making PGIS a more efficient catalyst for the 15-HPETE reaction.Keywords prostacyclin synthase; thromboxane synthase; cytochrome P450; peroxide bond cleavage; homolytic; heterolytic; hydroperoxides; epoxyalcohol Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) are members of the cytochrome P450 superfamily, which uses heme as the prosthetic group and has a cysteine thiolate proximal ligand [1,2]. PGIS and TXAS convert prostaglandin H 2 (PGH 2 ) to prostacyclin (PGI 2 ) and thromboxane A 2 (TXA 2 ), respectively. PGI 2 inhibits platelet activation and aggregation and induces vasodilation, whereas TXA 2 stimulates platelet secretion, aggregation and vasoconstriction [3]. Both PGI 2 and TXA 2 are rapidly hydrolyzed To whom correspondence should be addressed: Lee-Ho Wang Division of Hematology Department of Internal Medicine 6431 Fannin Houston, TX 77030 Tel: 713-500-6794 Fax: 713-500-6810 e-mail: lee-ho.wang@uth.tmc.edu 1 The abbreviations used are: PGIS, prostacyclin synthase; PGI 2 , prostacyclin; PGHS, prostaglandin H synthase; TXAS, thromboxane synthase; TXA 2 , thromboxane A 2 ; 8,11-eicosatrienoic acid; ESI/MS, electron-spray ionization/ mass spectrometry; EPR, electron paramagnetic resonance; SVD, singular value decomposition.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Hecker and Ullrich proposed a caged radical mechanism for TXAS ...

Prostacyclin synthase (PGIS) is a membrane-bound class III cytochrome P450 that catalyzes an isomerization of prostaglandin H 2 , an endoperoxide, to prostacyclin. We report here the characterization of the PGIS intermediates in reactions with other peroxides, peracetic acid (PA), and iodosylbenzene. Rapid-scan stopped-flow experiments revealed an intermediate with an absorption spectrum similar to that of compound ES (Cpd ES), which is an oxo-ferryl (Fe(IV)=O) plus a protein-derived radical. Cpd ES, formed upon reaction with PA, has an X-band (9 GHz) EPR signal of g = 2.0047 and a half-saturation power, P 1/2 , of 0.73 mW. High-field (130 GHz) EPR reveals the presence of two species of tyrosyl radicals in Cpd ES with their g-tensor components (g x , g y , g z ) of 2.00970, 2.00433, 2.00211 and 2.00700, 2.00433, 2.00211 at a 1:2 ratio, indicating that one is involved in hydrogen bonding and the other is not. The line width of the g = 2 signal becomes narrower, while its P 1/2 value becomes smaller as the reaction proceeds, indicating migration of the unpaired electron to an alternative site. The rate of electron migration (~0.2 s −1 ) is similar to that of heme bleaching, suggesting the migration is associated with the enzymatic inactivation. Moreover, a g = 6 signal that is presumably a high-spin ferric species emerges after the appearance of the amino acid radical and subsequently decays at a rate comparable to that of enzymatic inactivation. This loss of the g = 6 species thus likely indicates another pathway leading to enzymatic inactivation. The inactivation, however, was prevented by the exogenous reductant guaiacol. The studies of PGIS with PA described herein provide a mechanistic model of a peroxidase reaction catalyzed by the class III cytochromes P450.Cytochromes P450 (P450) 1 play important roles in physiological processes, pharmaceutical metabolism, and catalysis of a variety of reactions, such as hydroxylations, epoxidations, Nand O-dealkylations, and isomerizations. P450 enzymes contain a low-spin ferric heme with a cysteinate residue as the proximal ligand (1). Binding of the substrate often induces the heme to convert to high spin with detachment of the distal ligand and an increase of the redox