Inefficient spin trapping of superoxide in the presence of nitric-oxide: implications for studies on nitric-oxide synthase uncoupling

Uncoupling of nitric-oxide synthase (NOS) by deficiency of the substrate L-arginine or the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) is known to generate the reactive oxygen species H2O2 and superoxide. Discrimination between these two compounds is usually achieved by spin trapping of superoxide. We measured superoxide formation by uncoupled rat neuronal NOS, which contained one equivalent of tightly bound BH4 per dimer, using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap. As expected, the Ca2+-stimulated enzyme exhibited reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity that was accompanied by generation of superoxide and H2O2 in the absence of added L-arginine and BH4. Addition of BH4 (10 microM) did not significantly affect the rate of H2O2 formation but almost completely inhibited the apparent formation of superoxide, suggesting direct formation of H2O2. Although L-arginine (0.1 mM) increased the rate of NADPH oxidation about two-fold, the substrate largely attenuated apparent formation of both superoxide and H2O2, indicating that the spin trap did not efficiently outcompete the reaction between NO and superoxide. The efficiency of DEPMPO to scavenge superoxide in the presence of NO was studied by measuring free NO with a Clark-type electrode under conditions of NO/superoxide cogeneration. Neuronal NOS half-saturated with BH4 and the donor compound 3-morpholinosydnonimine (SIN-1) were used as enzymatic and nonenzymatic sources of NO/superoxide, respectively. Neither of the two systems gave rise to considerable NO signals in the presence of 50-100 mM DEPMPO, and even at 400 mM the spin trap uncovered less than 50% of the NO release that was detectable in the presence of 5000 U/ml superoxide dismutase. These results indicate that DEPMPO and all other currently available superoxide spin traps do not efficiently outcompete the reaction with NO. In addition, the similar behavior of nNOS and SIN-1 provides further evidence for NO as initial product of the NOS reaction.     

Pathogenetic role of eNOS uncoupling in cardiopulmonary disorders

The homodimeric flavohemeprotein endothelial nitric oxide synthase (eNOS) oxidizes l-arginine to l-citrulline and nitric oxide (NO), which acutely vasodilates blood vessels and inhibits platelet aggregation. Chronically, eNOS has a major role in the regulation of blood pressure and prevention of atherosclerosis by decreasing leukocyte adhesion and smooth muscle proliferation. However, a disturbed vascular redox balance results in eNOS damage and uncoupling of oxygen activation from l-arginine conversion. Uncoupled eNOS monomerizes and generates reactive oxygen species (ROS) rather than NO. Indeed, eNOS uncoupling has been suggested as one of the main pathomechanisms in a broad range of cardiovascular and pulmonary disorders such as atherosclerosis, ventricular remodeling, and pulmonary hypertension. Therefore, modulating uncoupled eNOS, in particular eNOS-dependent ROS generation, is an attractive therapeutic approach to preventing and/or treating cardiopulmonary disorders, including protective effects during cardiothoracic surgery. This review provides a comprehensive overview of the pathogenetic role of uncoupled eNOS in both cardiovascular and pulmonary disorders. In addition, the related therapeutic possibilities such as supplementation with the eNOS substrate l-arginine, volatile NO, and direct NO donors as well as eNOS modulators such as the eNOS cofactor tetrahydrobiopterin and folic acid are discussed in detail.     

Superoxide dismutase and catalase are required to detect (.-)NO from both coupled and uncoupled neuronal no synthase

Despite numerous approaches to measuring nitric oxide ((.-)NO) formation from purified NO synthase (NOS), it is still not clear whether (.-)NO is a direct or indirect product of the NO synthase reaction. The direct detection of catalytically formed (.-)NO is complicated by side reactions with reactive oxide species like H(2)O(2) and superoxide. The aim of the present study was therefore to reinvestigate these reactions both electrochemically and by chemiluminescence detection with particular emphasis on the requirement for cofactors and their interference with (.-)NO detection. Flavins were found to generate large amounts of H(2)O(2) and were therefore excluded from subsequent incubations. Under conditions of both coupled and uncoupled catalysis, SOD was absolutely required to detect (.-)NO from NOS. H(2)O(2) formation took place also in the presence of SOD and gave a smaller yet significant interfering signal. Similar data were obtained when the proposed intermediate N(omega)-hydroxy-l-arginine was utilized as substrate. In conclusion, standard Clark-type ()NO electrodes are cross-sensitive to H(2)O(2) and therefore both SOD and catalase are absolutely required to specifically detect (.-)NO from NOS.     

L-arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa

Sperm capacitation and acrosome reaction are essential for fertilization and they are considered as part of an oxidative process involving superoxide and hydrogen peroxide. In human spermatozoa, the amino acid L-arginine is a substrate for the nitric oxide synthase (NOS) producing nitric oxide (NO*), a reactive molecule that participates in capacitation as well as in acrosome reaction. L-arginine plays an important role in the physiology of spermatozoa and has been shown to enhance their metabolism and maintain their motility. Moreover, L-arginine has a protective effect on spermatozoa against the sperm plasma membrane lipid peroxidation. In this paper, we have presented, for the first time, the effect of L-arginine on cryopreserved bovine sperm capacitation and acrosome reaction and the possible participation of NOS in both processes. Frozen-thawed bovine spermatozoa have been incubated in TALP medium with different concentrations of L-arginine and the percentages of capacitated and acrosome reacted spermatozoa have been determined. L-arginine induced both capacitation and acrosome reaction. NO* produced by L-arginine has been inhibited or inactivated using NOS inhibitors or NO* scavengers in the incubation medium, respectively. Thus, the effect of NOS inhibitors and NO* scavengers in capacitated and non-capacitated spermatozoa treated with L-arginine has also been monitored. The data presented suggest the participation of NO*, produced by a sperm NOS, in cryopreseved bovine sperm capacitation and acrosome reaction.     

Redox function of tetrahydrobiopterin and effect of L-arginine on oxygen binding in endothelial nitric oxide synthase

Tetrahydrobiopterin (BH(4)), not dihydrobiopterin or biopterin, is a critical element required for NO formation by nitric oxide synthase (NOS). To elucidate how BH(4) affects eNOS activity, we have investigated BH(4) redox functions in the endothelial NOS (eNOS). Redox-state changes of BH(4) in eNOS were examined by chemical quench/HPLC analysis during the autoinactivation of eNOS using oxyhemoglobin oxidation assay for NO formation at room temperature. Loss of NO formation activity linearly correlated with BH(4) oxidation, and was recovered by overnight incubation with fresh BH(4). Thus, thiol reagents commonly added to NOS enzyme preparations, such as dithiothreitol and beta-mercaptoethanol, probably preserve enzyme activity by preventing BH(4) oxidation. It has been shown that conversion of L-arginine to N-hydroxy-L-arginine in the first step of NOS catalysis requires two reducing equivalents. The first electron that reduces ferric to the ferrous heme is derived from flavin oxidation. The issue of whether BH(4) supplies the second reducing equivalent in the monooxygenation of eNOS was investigated by rapid-scan stopped-flow and rapid-freeze-quench EPR kinetic measurements. In the presence of L-arginine, oxygen binding kinetics to ferrous eNOS or to the ferrous eNOS oxygenase domain (eNOS(ox)) followed a sequential mechanism: Fe(II) <--> Fe(II)O(2) --> Fe(III) + O(2)(-). Without L-arginine, little accumulation of the Fe(II)O(2) intermediate occurred and essentially a direct optical transition from the Fe(II) form to the Fe(III) form was observed. Stabilization of the Fe(II)O(2) intermediate by L-arginine has been established convincingly. On the other hand, BH(4) did not have significant effects on the oxygen binding and decay of the oxyferrous intermediate of the eNOS or eNOS oxygenase domain. Rapid-freeze-quench EPR kinetic measurements in the presence of L-arginine showed a direct correlation between BH(4) radical formation and decay of the Fe(II)O(2) intermediate, indicating that BH(4) indeed supplies the second electron for L-arginine monooxygenation in eNOS.     

Zinc content of Escherichia coli-expressed constitutive isoforms of nitric-oxide synthase. Enzymatic activity and effect of pterin.

Recently, we obtained x-ray crystallographic data showing the presence of a ZnS4 center in the structure of Escherichia coli-expressed bovine endothelial nitric-oxide synthase (eNOS) and rat neuronal nitric-oxide synthase (nNOS). The zinc atom is coordinated by two CXXXXC motifs, one motif being contributed by each NOS monomer (cysteine 326 through cysteine 331 in rat nNOS). Mutation of the nNOS cysteine 331 to alanine (C331A) results in the loss of NO. synthetic activity and also results in an inability to bind zinc efficiently. Although prolonged incubation of the C331A mutant of nNOS with high concentrations of L-arginine results in a catalytically active enzyme, zinc binding is not restored. In this study, we investigate the zinc stoichiometry in wild-type nNOS and eNOS, as well as in the C331A-mutated nNOS, using a chelation assay and electrothermal vaporization-inductively coupled plasma-mass spectrometry. The data reveal an approximate 2:1 stoichiometry of heme to zinc in (6R)-5,6,7,8-tetrahydro-L-biopterin-replete, wild-type nNOS and eNOS and show that the reactivated C331A mutant of nNOS has a limited ability to bind zinc. The present study substantiates that the zinc in NOS is structural rather than catalytic and is important for maintaining optimally functional, enzymatically active, constitutive NOSs.     

eNOS function is developmentally regulated: uncoupling of eNOS occurs postnatally

At birth, the transition to gas breathing requires the function of endothelial vasoactive agents. We investigated the function of endothelial nitric oxide synthase (eNOS) in pulmonary artery (PA) vessels and endothelial cells isolated from fetal and young (4-wk) sheep. We found greater relaxations to the NOS activator A-23187 in 4-wk-old compared with fetal vessels and that the NOS inhibitor nitro-L-arginine blocked relaxations in both groups. Relaxations in 4-wk vessels were not blocked by an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, but were partially blocked by catalase. We therefore hypothesized that activation of eNOS produced reactive oxygen species in 4-wk but not fetal PA. To address this question, we studied NO and superoxide production by endothelial cells at baseline and following NOS stimulation with A-23187, VEGF, and laminar shear stress. Stimulation of NOS induced phosphorylation at serine 1177, and this event correlated with an increase in NO production in both ages. Upon stimulation of eNOS, fetal PA endothelial cells (PAEC) produced only NO. In contrast 4-wk-old PAEC produced superoxide in addition to NO. Superoxide production was blocked by L-NAME but not by apocynin (an NADPH oxidase inhibitor). L-Arginine increased NO production in both cell types but did not block superoxide production. Heat shock protein 90/eNOS association increased upon stimulation and did not change with developmental age. Cellular levels of total and reduced biopterin were higher in fetal vs. 4-wk cells. Sepiapterin [a tetrahydrobiopterin (BH4) precursor] increased basal and stimulated NO levels and completely blocked superoxide production. We conclude that the normal function of eNOS becomes uncoupled after birth, leading to a developmental adaptation of the pulmonary vascular system to produce oxygen species other than NO. We speculate this may be related to cellular production and/or maintenance of BH4 levels.     

Three different oxygen-induced radical species in endothelial nitric-oxide synthase oxygenase domain under regulation by L-arginine and tetrahydrobiopterin

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 L-arginine 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. BH(4)-free eNOS(ox) produced the superoxide radical very efficiently in the absence of L-arginine. L-Arginine decreased the formation rate of superoxide by an order of magnitude but not its final level or EPR line shape. For BH(4)-containing eNOS(ox), only a stoichiometric amount of BH(4) radical was produced in the presence of L-arginine, but in its absence a new radical was obtained. This new radical could be either a peroxyl radical of BH(4) or an amino acid radical was in the vicinity of the heme. Formation of this new radical is very rapid, >150 s(-1), and it was subsequently converted to a BH(4) radical. The trapping of the superoxide radical by cytochrome c in the reaction of BH(4)(-) eNOS(ox) exhibited a limiting rate of approximately 15 s(-1), the time for the superoxide radical to leave the heme pocket and reach the protein surface; this reveals a general problem of the regular spin-trapping method in determining radical formation kinetics. Cytochrome c failed to trap the new radical species. Together with other EPR characteristics, our data strongly support the conclusion that this new radical is not a superoxide radical or a mixture of superoxide and biopterin radicals. Our study points out distinct roles of BH(4) and L-arginine in regulating eNOS radical intermediates. BH(4) prevented superoxide formation by chemical conversions of the Fe(II)O(2) intermediate, and l-arginine delayed superoxide formation by electronic interaction with the heme-bound oxygen.     

Reactivity of the heme-dioxygen complex of the inducible nitric oxide synthase in the presence of alternative substrates

Single turnover reactions of the inducible nitric oxide synthase oxygenase domain (iNOSoxy) in the presence of several non alpha-amino acid N-hydroxyguanidines and guanidines were studied by stopped-flow visible spectroscopy, and compared with reactions using the native substrates L-arginine (L-arg) or N(omega)-hydroxy-L-arginine (NOHA). In experiments containing dihydrobiopterin, a catalytically incompetent pterin, and each of the studied substrates, L-arg, butylguanidine (BuGua), para-fluorophenylguanidine (FPhGua), NOHA, N-butyl- and N-(para-fluorophenyl)-N'-hydroxyguanidines (BuNOHG and FPhNOHG), the formation of a iron(II) heme-dioxygen intermediate (Fe(II)O2) was always observed. The Fe(II)O2 species then decayed to iron(III) iNOSoxy at rates that were dependent on the nature of the substrate. Identical reactions containing the catalytically competent cofactor tetrahydrobiopterin (BH4), iNOSoxy and the three N-hydroxyguanidines, all exhibited an initial formation of an Fe(II)O2 species that was successively converted to an Fe(III)NO complex and eventually to high-spin iron(III) iNOSoxy. The formation and decay kinetics of the Fe(III)NO complex did not vary greatly as a function of the N-hydroxyguanidine structure, but the formation of Fe(III)NO was substoichiometric in the cases of BuNOHG and FPhNOHG. Reactions between BH4-containing iNOSoxy and BuGua exhibited kinetics similar to those of the corresponding reaction with L-arginine, with formation of an Fe(II)O2 intermediate that was directly converted to high-spin iron(III) iNOSoxy. In contrast, no Fe(II)O2 intermediate was observed in the reaction of BH4-containing iNOSoxy and FPhGua. Multi-turnover reaction of iNOS with FPhGua did not lead to formation of NO or to hydroxylation of the substrate, contrary to reactions with BuGua or L-arg. Our results reveal how different structural and chemical properties of NOS substrate analogues can impact on the kinetics and reactivity of the Fe(II)O2 intermediate, and support an important role for substrate pKa during NOS oxygen activation.     

Superoxide induces endothelial nitric-oxide synthase protein thiyl radical formation, a novel mechanism regulating eNOS function and coupling

An increase in production of reactive oxygen species resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases, and these reactive oxygen species can oxidize cellular macromolecules. Protein thiols are critical reducing equivalents that maintain cellular redox state and are primary targets for oxidative modification. We demonstrate endothelial NOS (eNOS) oxidant-induced protein thiyl radical formation from tetrahydrobiopterin-free enzyme or following exposure to exogenous superoxide using immunoblotting, immunostaining, and mass spectrometry. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) followed by immunoblotting using an anti-DMPO antibody demonstrated the formation of eNOS protein radicals, which were abolished by superoxide dismutase and L-NAME, indicating that protein radical formation was due to superoxide generation from the eNOS heme. With tetrahydrobiopterin-reconstituted eNOS, eNOS protein radical formation was completely inhibited. Using mass spectrometric and mutagenesis analysis, we identified Cys-908 as the residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS, leading to increased superoxide generation. Protein thiyl radical formation leads to oxidation or modification of cysteine with either disulfide bond formation or S-glutathionylation, which induces eNOS uncoupling. Furthermore, in endothelial cells treated with menadione to trigger cellular superoxide generation, eNOS protein radical formation, as visualized with confocal microscopy, was increased, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Thus, eNOS protein radical formation provides the basis for a mechanism of superoxide-directed regulation of eNOS, involving thiol oxidation, defining a unique pathway for the redox regulation of cardiovascular function.     

Involvement of the perferryl complex of nitric oxide synthase in the catalysis of secondary free radical formation

Neuronal nitric oxide synthase (NOS I) has been shown to generate nitric oxide (NO*) and superoxide (O(2)* during enzymatic cycling, and the ratio of each free radical is dependent upon the concentration of L-arginine. Using spin trapping and electron paramagnetic resonance spectroscopy, we detected alpha-hydroxyethyl radical (CH(3)*CHOH), produced during the NOS I metabolism of ethanol (EtOH). The generation of CH(3)*CHOH by NOS I was found to be Ca(2+)/calmodulin dependent. Superoxide dismutase prevented CH(3)*CHOH formation in the absence of L-arginine. However, in the presence of L-arginine, the production of CH(3)*CHOH was independent of O(2)* but dependent upon the concentration of L-arginine. Formation of CH(3)*CHOH was inhibited by substituting D-arginine for L-arginine, or inclusion of the NOS inhibitors N(G)-nitro-L-arginine methyl ester, N(G)-monomethyl-L-arginine and the heme blocker, sodium cyanide. The addition of potassium hydrogen persulfate to NOS I, generating the perferryl complex (NOS-[Fe(5+)=O](3+)) in the absence of oxygen and Ca(2+)/calmodulin, and EtOH resulted in the formation of CH(3)*CHOH. NOS I was found to produce the corresponding alpha-hydroxyalkyl radical from 1-propanol and 2-propanol, but not from 2-methyl-2-propanol. Data demonstrated that the perferryl complex of NOS I in the presence of L-arginine was responsible for catalyses of these secondary reactions.     

Oxygen-induced radical intermediates in the nNOS oxygenase domain regulated by L-arginine, tetrahydrobiopterin, and thiol

Fully coupled nitric oxide synthase (NOS) catalyzes formation of nitric oxide (NO), l-citrulline, NADP+, and water from l-arginine, NADPH, and oxygen. Uncoupled or partially coupled NOS catalyzes the synthesis of reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite, depending on the availability of cofactor tetrahydrobiopterin (BH4) and l-arginine during catalysis. We identified three distinct oxygen-induced radical intermediates in the ferrous endothelial NOS oxygenase domain (eNOSox) with or without BH4 and/or l-arginine [Berka, V., Wu, G., Yeh, H. C., Palmer, G., and Tsai, A.-L. (2004) J. Biol. Chem. 279, 32243-32251]. The effects of BH4 and l-arginine on the oxygen-induced radical intermediates in the isolated neuronal NOS oxygenase domain (nNOSox) have been similarly investigated by single-turnover stopped-flow and rapid-freeze quench EPR kinetic measurements in the presence or absence of dithiothreitol (DTT). Like for eNOSox, we found different radical intermediates in the reaction of ferrous nNOSox with oxygen. (1) nNOSox (without BH4 or l-Arg) produces superoxide in the presence or absence of DTT. (2) nNOSox (with BH4 and l-Arg) yields a typical BH4 radical in a manner independent of DTT. (3) nNOSox (with BH4 and without l-Arg) yields a new radical. Without DTT, EPR showed a mixture of superoxide and biopterin radicals. With DTT, a new approximately 75 G wide radical EPR was observed, different from the radical formed by eNOSox. (4) The presence of only l-arginine in nNOSox (without BH4 but with l-Arg) caused conversion of approximately 70% of superoxide radical to a novel radical, explaining how l-arginine decreases the level of superoxide production in nNOSox (without BH4 but with l-Arg). The regulatory role of l-arginine in nNOS is thus very different from that in eNOS where substrate was only to decrease the rate of formation of superoxide but not the total amount of radical. The role of DTT is also different. DTT prevents oxidation of BH4 in both isoforms, but in nNOS, DTT also inhibits oxidation of two key cysteines in nNOSox to prevent the loss of substrate binding. This new role of thiol found only for nNOS may be significant in neurodegenerative diseases.     

Redox properties of human endothelial nitric-oxide synthase oxygenase and reductase domains purified from yeast expression system

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 rapid-freeze 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.     

Characterization of interactions among the heme center, tetrahydrobiopterin, and L-arginine binding sites of ferric eNOS using imidazole, cyanide, and nitric oxide as probes

Endothelial nitric oxide synthase (eNOS) is a self-sufficient P450-like enzyme. A P450 reductase domain is tethered to an oxygenase domain containing the heme, the substrate (L-arginine) binding site, and a cofactor, tetrahydrobiopterin (BH(4)). This "triad", located at the distal heme pocket, is the center of oxygen activation and enzyme catalysis. To probe the relationships among these three components, we examined the binding kinetics of three different small heme ligands in the presence and absence of either L-arginine, BH(4), or both. Imidazole binding was strictly competitive with L-arginine, indicating a domain overlap. BH(4) had no obvious effect on imidazole binding but slightly increased the k(on) for L-arginine. L-Arginine decreased the k(on) and k(off) for cyanide by two orders, indicating a "kinetic obstruction" mechanism. BH(4) slightly enhanced cyanide binding. Nitric oxide (NO) binding kinetics were more complex. Increasing the L-arginine concentration decreased the NO binding affinity at equilibrium. In both BH(4)-abundant and BH(4)-deficient eNOS, half of the NO binding sites showed a sizable decrease of the binding rate by L-arginine, with the rate of NO binding at the other half of the sites remaining essentially unaltered by L-arginine, implying that the two heme centers in the eNOS dimer are functionally distinct.     

Janus-faced role of endothelial NO synthase in vascular disease: uncoupling of oxygen reduction from NO synthesis and its pharmacological reversal

Endothelial NO synthase (eNOS) is the predominant enzyme responsible for vascular NO synthesis. A functional eNOS transfers electrons from NADPH to its heme center, where L-arginine is oxidized to L-citrulline and NO. Common conditions predisposing to atherosclerosis, such as hypertension, hypercholesterolemia, diabetes mellitus and smoking, are associated with enhanced production of reactive oxygen species (ROS) and reduced amounts of bioactive NO in the vessel wall. NADPH oxidases represent major sources of ROS in cardiovascular pathophysiology. NADPH oxidase-derived superoxide avidly interacts with eNOS-derived NO to form peroxynitrite (ONOO(-)), which oxidizes the essential NOS cofactor (6R-)5,6,7,8-tetrahydrobiopterin (BH(4)). As a consequence, oxygen reduction uncouples from NO synthesis, thereby rendering NOS to a superoxide-producing pro-atherosclerotic enzyme. Supplementation with BH(4) corrects eNOS dysfunction in several animal models and in patients. Administration of high local doses of the antioxidant L-ascorbic acid (vitamin C) improves endothelial function, whereas large-scale clinical trials do not support a strong role for oral vitamin C and/or E in reducing cardiovascular disease. Statins, angiotensin-converting enzyme inhibitors and AT1 receptor blockers have the potential of reducing vascular oxidative stress. Finally, novel approaches are being tested to block pathways leading to oxidative stress (e.g. protein kinase C) or to upregulate antioxidant enzymes.     

Use of kinetic isotope effects to delineate the role of phenylalanine 87 in P450(BM-3)

The substrate oxidation rates of P450(BM-3) are unparalleled in the cytochrome P450 (CYP) superfamily of enzymes. Furthermore, the bacterial enzyme, originating from Bacillus megaterium, has been used repeatedly as a model to study the metabolism of mammalian P450s. A specific example is presented where studying P450(BM-3) substrate dynamics can define important enzyme-substrate characteristics, which may be useful in modeling omega-hydroxylation seen in mammalian P450s. In addition, if the reactive species responsible for metabolism can be controlled to produce specific products this enzyme could be a useful biocatalyst. Based on crystal structures and the fact that the P450(BM-3) F87A mutant produces a large isotope in contrast to the native enzyme, we propose that phenylalanine 87 is responsible for hindering substrate access to the active oxygen species for nonnative substrates. Using kinetic isotopes and two aromatic substrates, p-xylene and 4,4'-dimethylbiphenyl, the role phenylalanine 87 plays in active-site dynamics is characterized. The intrinsic KIE is 7.3 +/- 2 for wtP450(BM-3) metabolism of p-xylene. In addition, stoichiometry differences were measured with the native and mutant enzyme and 4,4'-dimethylbiphenyl. The results show a more highly coupled substrate/NADPH ratio in the mutant than in the wtP450(BM-3).     

Asymmetric Dimethylarginine (ADMA) and other Endogenous Nitric Oxide Synthase (NOS) Inhibitors as an Important Cause of Vascular Insulin Resistance

Asymmetric dimethylarginine (ADMA) and NG-monomethyl- L-arginine ( L-NMMA) are important endogenous endothelial nitric oxide synthase (eNOS) inhibitors. Studies have shown that patients with insulin resistance have elevated plasma levels of ADMA. Moreover, ADMA levels have a prognostic value on long-term outcome of patients with coronary artery disease. Insulin resistance, a disorder associated to inadequate biological responsiveness to the actions of exogenous or endogenous insulin, is a metabolic condition, which exists in patients with cardiovascular diseases. This disorder affects the functional balance of vascular endothelium via changes of nitric oxide (NO) metabolism. Nitric oxide is produced in endothelial cells from the substrate L-arginine via eNOS. Elevated ADMA levels cause eNOS uncoupling, a mechanism which leads to decreased NO bioavailability and increased production of hydrogen peroxide. According to clinical studies, the administration of L-arginine to patients with high ADMA levels improves NO synthesis by antagonizing the deleterious effect of ADMA on eNOS function, although in specific populations such as diabetes mellitus, this might even been harmful. More studies are required in order to certify the role of NOS inhibitors in insulin resistance and endothelial dysfunction. It is still difficult to say whether increased ADMA levels in certain populations is only a reason or the result of the molecular alterations, which take place in vascular disease states.     

Synthesis and evaluation of trans 3,4-cyclopropyl L-arginine analogues as isoform selective inhibitors of nitric oxide synthase

Four optically pure conformationally restricted L-arginine analogues syn- 1 and anti- 2 trans-3,4-cyclopropyl L-arginine, and syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were synthesized. These compounds were tested as potential inhibitors against the three isoforms of nitric oxide synthase (NOS). Compound 1 was determined to be a poor substrate of NOS, while compound 2 was determined to be a poor mixed type inhibitor and did not exhibit any isoform selectivity. Syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were found to be competitive inhibitors of NOS. These compounds were time dependent inhibitors of inducible NOS (iNOS), but not of neuronal NOS (nNOS) or endothelial NOS (eNOS). Compound 3 was 10- to 100-fold more potent an inhibitor than 4, exhibited a 5-fold increase in nNOS/iNOS and eNOS/iNOS selectivity over 4, and displayed tight binding characteristics against iNOS. These results indicate that the relative configuration of the cyclopropyl ring in the L-arginine analogues significantly affects their inhibitory potential and NOS isoform selectivity.