CoQ10 increases mitochondrial mass and polarization, ATP and Oct4 potency levels, and bovine oocyte MII during IVM while decreasing AMPK activity and oocyte death.

PURPOSE: We tested whether mitochondrial electron transport chain electron carrier coenzyme Q10 (CoQ10) increases ATP during bovine IVM and increases %M2 oocytes, mitochondrial polarization/mass, and Oct4, and decreases pAMPK and oocyte death. METHODS: Bovine oocytes were aspirated from ovaries and cultured in IVM media for 24 h with 0, 20, 40, or 60 µM CoQ10. Oocytes were assayed for ATP by luciferase-based luminescence. Oocyte micrographs were quantitated for Oct4, pAMPK (i.e., activity), polarization by JC1 staining, and mitochondrial mass by MitoTracker Green staining. RESULTS: CoQ10 at 40 µM was optimal. Oocytes at 40 µM enabled 1.9-fold more ATP than 0 µM CoQ10. There was 4.3-fold less oocyte death, 1.7-fold more mitochondrial charge polarization, and 3.1-fold more mitochondrial mass at 40 µM than at 0 µM CoQ10. Increased ATP was associated with 2.2-fold lower AMPK thr172P activation and 2.1-fold higher nuclear Oct4 stemness/potency protein at 40 µM than at 0 µM CoQ10. CoQ10 is hydrophobic, and at all doses, 50% was lost from media into oil by ~ 12 h. Replenishing CoQ10 at 12 h did not significantly diminish dead oocytes. CONCLUSIONS: The data suggest that CoQ10 improves mitochondrial function in IVM where unwanted stress, higher AMPK activity, and Oct4 potency loss are induced.

Uterine fibroids are characterized by an impaired antioxidant cellular system: potential role of hypoxia in the pathophysiology of uterine fibroids.

PURPOSE: Fibroids are the most common smooth muscle overgrowth in women. This study determined the expression and the effect of hypoxia on two potent antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) on human fibroid cells. METHODS: Immortalized human leiomyoma (fibroid) and myometrial cells were subjected to hypoxia (2 % O2, 24 h). Total RNA and cell homogenate were obtained from control and treated cells; CAT and SOD mRNA and activity levels were determined by real-time RT-PCR and ELISA, respectively. RESULTS: Fibroid cells have significantly lower antioxidant enzymes, SOD and CAT mRNA and activity levels than normal myometrial cells (p < 0.05). Hypoxia treatment significantly increased SOD activity in myometrial cells while significantly decreasing CAT activity in fibroid cells (p < 0.05). There was no significant difference in CAT mRNA levels or activity in response to hypoxia in myometrial cells. Also, there was no significant difference in SOD mRNA levels in response to hypoxia in myometrial cells. CONCLUSION: This is the first report to show that uterine fibroids are characterized by an impaired antioxidant cellular enzymatic system. More importantly, our results indicate a role for hypoxia in the modulation of the balance of those enzymes in fibroid and myometrial cells. Collectively, these results shed light on the pathophysiology of fibroids thereby providing potential targets for novel fibroid treatment.

Analysis of the mechanism by which melatonin inhibits human eosinophil peroxidase.

BACKGROUND AND PURPOSE: Eosinophil peroxidase (EPO) catalyses the formation of oxidants implicated in the pathogenesis of various respiratory diseases including allergy and asthma. Mechanisms for inhibiting EPO, once released, are poorly understood. The aim of this work is to determine the mechanisms by which melatonin, a hormone produced in the brain by the pineal gland, inhibits the catalytic activity of EPO. EXPERIMENTAL APPROACH: We utilized H2O2-selective electrode and direct rapid kinetic measurements to determine the pathways by which melatonin inhibits human EPO. KEY RESULTS: In the presence of plasma levels of bromide (Br-), melatonin inactivates EPO at two different points in the classic peroxidase cycle. First, it binds to EPO and forms an inactive complex, melatonin-EPO-Br, which restricts access of H2O2 to the catalytic site of the oxidation enzyme. Second, melatonin competes with Br- and switches the reaction from a two electron (2e-) to a one electron (1e-) pathway allowing the enzyme to function with catalase-like activity. Melatonin is a bulky molecule and binds to the entrance of the EPO haem pocket (regulatory sites). Furthermore, Br- seems to enhance the affinity of this binding. In the absence of Br-, melatonin accelerated formation of EPO Compound II and its decay by serving as a 1e- substrate for EPO Compounds I and II. CONCLUSIONS AND IMPLICATIONS: The interplay between EPO and melatonin may have a broader implication in the function of several biological systems. This dual regulation by melatonin is unique and represents a new mechanism for melatonin to control EPO and its downstream inflammatory pathways.

Nitric oxide synthesis in the lung. Regulation by oxygen through a kinetic mechanism.

In this study, we show that oxygen regulates nitric oxide (NO) levels through effects on NO synthase (NOS) enzyme kinetics. Initially, NO synthesis in the static lung was measured in bronchiolar gases during an expiratory breath-hold in normal individuals. NO accumulated exponentially to a plateau, indicating balance between NO production and consumption in the lung. Detection of NO2-, NO3-, and S-nitrosothiols in lung epithelial lining fluids confirmed NO consumption by chemical reactions in the lung. Interestingly, alveolar gas NO (estimated from bronchiolar gases at end-expiration) was near zero, suggesting NO in exhaled gases is not derived from circulatory/systemic sources. Dynamic NO levels during tidal breathing in different airway regions (mouth, trachea, bronchus, and bronchiole) were similar. However, in individuals breathing varying levels of inspired oxygen, dynamic NO levels were notably dependent on O2 concentration in the hypoxic range (KmO2 190 microM). Purified NOS type II enzyme activity in vitro was similarly dependent on molecular oxygen levels (KmO2 135 microM), revealing a means by which oxygen concentration affects NO levels in vivo. Based upon these results, we propose that NOS II is a mediator of the vascular response to oxygen in the lung, because its KmO2 allows generation of NO in proportion to the inspired oxygen concentration throughout the physiologic range.

Formation of nitric oxide-derived oxidants by myeloperoxidase in monocytes: pathways for monocyte-mediated protein nitration and lipid peroxidation In vivo.

Protein nitration and lipid peroxidation are implicated in the pathogenesis of atherosclerosis; however, neither the cellular mediators nor the reaction pathways for these events in vivo are established. In the present study, we examined the chemical pathways available to monocytes for generating reactive nitrogen species and explored their potential contribution to the protein nitration and lipid peroxidation of biological targets. Isolated human monocytes activated in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide ((*)NO) metabolism, nitrate apolipoprotein B-100 tyrosine residues and initiate LDL lipid peroxidation. LDL nitration (assessed by gas chromatography-mass spectrometry quantification of nitrotyrosine) and lipid peroxidation (assessed by high-performance liquid chromatography with online tandem mass spectrometric quantification of distinct products) required cell activation and NO(2)(-); occurred in the presence of metal chelators, superoxide dismutase (SOD), and scavengers of hypohalous acids; and was blocked by myeloperoxidase (MPO) inhibitors and catalase. Monocytes activated in the presence of the exogenous (*)NO generator PAPA NONOate (Z-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2- diolate) promoted LDL protein nitration and lipid peroxidation by a combination of pathways. At low rates of (*)NO flux, both protein nitration and lipid peroxidation were inhibited by catalase and peroxidase inhibitors but not SOD, suggesting a role for MPO. As rates of (*)NO flux increased, both nitrotyrosine formation and 9-hydroxy-10,12-octadecadienoate/9-hydroperoxy-10,12-octadecadieno ic acid production by monocytes became insensitive to the presence of catalase or peroxidase inhibitors, but they were increasingly inhibited by SOD and methionine, suggesting a role for peroxynitrite. Collectively, these results demonstrate that monocytes use distinct mechanisms for generating (*)NO-derived oxidants, and they identify MPO as a source of nitrating intermediates in monocytes.

Myeloperoxidase-generated oxidants and atherosclerosis.

Atherosclerosis is a chronic inflammatory process where oxidative damage within the artery wall is implicated in the pathogenesis of the disease. Mononuclear phagocytes, an inflammatory cell capable of generating a variety of oxidizing species, are early components of arterial lesions. Their normal functions include host defense and surveillance through regulated generation of diffusible radical species, reactive oxygen or nitrogen species, and HOCl (hypochlorous acid). However, under certain circumstances an excess of these oxidizing species can overwhelm local antioxidant defenses and lead to oxidant stress and oxidative tissue injury, processes implicated in the pathogenesis of atherosclerosis. This review focuses on oxidation reactions catalyzed by myeloperoxidase (MPO), an abundant heme protein secreted from activated phagocytes which is present in human atherosclerotic lesions. Over the past several years, significant evidence has accrued demonstrating that MPO is one pathway for protein and lipoprotein oxidation during the evolution of cardiovascular disease. Multiple distinct products of MPO are enriched in human atherosclerotic lesions and LDL recovered from human atheroma. However, the biological consequences of these MPO-catalyzed reactions in vivo are still unclear. Here we discuss evidence for the occurrence of MPO-catalyzed oxidation reactions in vivo and the potential role MPO plays in both normal host defenses and inflammatory diseases like atherosclerosis.

Nicotinamide Adenine Dinucleotide Phosphate Oxidase Expression Is Differentially Regulated to Favor a Pro-oxidant State That Contributes to Postoperative Adhesion Development.

We have previously reported that superoxide (O2•-) contributes to the development of postoperative adhesions. In this study, we determined whether O2•- generating nicotinamide adenine dinucleotide phosphate oxidase (NOX) is differentially expressed in normal peritoneal and adhesion fibroblasts and tissues. The NOX isoforms were measured utilizing Western blot, immunohistochemistry, high-performance liquid chromatography, and real-time reverse transcription polymerase chain reaction. Expression and activity of NOX were found to be significantly higher in adhesion tissues and cells than that in normal peritoneal tissues and cells (P < .05). Levels of NOX2, NOX4, NOX activating protein 1, DUOX1, p47phox, and p22phox messenger RNA increased in adhesion fibroblasts when compared to normal peritoneal and increased in response to hypoxia in normal peritoneal fibroblasts. Thus, adhesion fibroblasts are characterized by a unique NOX expression profile, which maintains a pro-oxidant state that may be responsible for the persistence of the adhesion phenotype. Decreasing the activity of NOX by targeting these isoforms may be beneficial for future therapeutic interventions of postoperative adhesions.

Regulation of inducible nitric oxide synthase in post-operative adhesions.

BACKGROUND: The deficiency of the inducible nitric oxide synthase (iNOS) substrate, L-arginine (L-Arg), the co-factor tetrahydrobiopterin (H4B) or molecular oxygen may lead to lower NO levels, which enhances the development of adhesion phenotype. METHODS: We utilized high-performance liquid chromatography (HPLC) and immunoprecipitation with nitrotyrosine antibody to determine the levels of H4B, citrulline and protein nitration in fibroblasts established from normal peritoneal and adhesion tissues. RESULTS: The level of H4B was dramatically attenuated in adhesion fibroblasts. The immunoprecipitation with nitrotyrosine antibody revealed higher protein nitration in adhesion compared with normal fibroblasts. There were higher accumulations of citrulline in adhesion fibroblasts as compared with normal fibroblasts. In addition, peritoneal fibroblasts treated with 2% oxygen for 24 h and implanted back into the peritoneal cavity of the rats exhibited marked increase in severity of adhesion as well as extensive distribution involving many sites and organs. CONCLUSIONS: Control of the catalytic activity of iNOS in adhesion fibroblasts may be because of subsaturating amounts of L-Arg and H4B which allow iNOS to generate a combination of reactive oxygen species in addition to NO, thereby influencing NO bioavailability and function.

Nitric oxide synthases reveal a role for calmodulin in controlling electron transfer.

Nitric oxide (NO) is synthesized within the immune, vascular, and nervous systems, where it acts as a wide-ranging mediator of mammalian physiology. The NO synthases (EC 1.14.13.39) isolated from neurons or endothelium are calmodulin dependent. Calmodulin binds reversibly to neuronal NO synthase in response to elevated Ca2+, triggering its NO production by an unknown mechanism. Here we show that calmodulin binding allows NADPH-derived electrons to pass onto the heme group of neuronal NO synthase. Calmodulin-triggered electron transfer to heme was independent of substrate binding, caused rapid enzymatic oxidation of NADPH in the presence of O2, and was required for NO synthesis. An NO synthase isolated from cytokine-induced macrophages that contains tightly bound calmodulin catalyzed spontaneous electron transfer to its heme, consistent with bound calmodulin also enabling electron transfer within this isoform. Together, these results provide a basis for how calmodulin may regulate NO synthesis. The ability of calmodulin to trigger electron transfer within an enzyme is unexpected and represents an additional function for calcium-binding proteins in biology.

Nitric oxide is a physiological substrate for mammalian peroxidases.

We now show that NO serves as a substrate for multiple members of the mammalian peroxidase superfamily under physiological conditions. Myeloperoxidase (MPO), eosinophil peroxidase, and lactoperoxidase all catalytically consumed NO in the presence of the co-substrate hydrogen peroxide (H(2)O(2)). Near identical rates of NO consumption by the peroxidases were observed in the presence versus absence of plasma levels of Cl(-). Although rates of NO consumption in buffer were accelerated in the presence of a superoxide-generating system, subsequent addition of catalytic levels of a model peroxidase, MPO, to NO-containing solutions resulted in the rapid acceleration of NO consumption. The interaction between NO and compounds I and II of MPO were further investigated during steady-state catalysis by stopped-flow kinetics. NO dramatically influenced the build-up, duration, and decay of steady-state levels of compound II, the rate-limiting intermediate in the classic peroxidase cycle, in both the presence and absence of Cl(-). Collectively, these results suggest that peroxidases may function as a catalytic sink for NO at sites of inflammation, influencing its bioavailability. They also support the potential existence of a complex and interdependent relationship between NO levels and the modulation of steady-state catalysis by peroxidases in vivo.

Interrogation of heme pocket environment of mammalian peroxidases with diatomic ligands.

Recent studies demonstrate that myeloperoxidase (MPO), eosinophil peroxidase (EPO), and lactoperoxidase (LPO), homologous members of the mammalian peroxidase superfamily, can all serve as catalysts for generating nitric oxide- (nitrogen monoxide, NO) derived oxidants. These enzymes contain heme prosthetic groups that are ligated through a histidine nitrogen and use H(2)O(2) as the electron acceptor in the catalysis of oxidative reactions. Here we show that heme reduction of these peroxidases results in distinct electronic and/or conformational changes in their heme pockets using a combination of rapid kinetics measurements, optical absorbance, and diatomic ligand binding studies. Addition of reducing agent to each peroxidase at ground state [Fe(III) state] causes immediate buildup of the corresponding Fe(II) complexes. Spectral changes indicate that two LPO-Fe(II) species are present in solution at equilibrium. Analyses of stopped-flow traces collected when EPO, MPO, or LPO solutions rapidly mixed with NO were accurately fit by single-exponential functions. Plots of the apparent rate constants as a function of NO concentration for all Fe(III) and Fe(II) forms were linear with positive intercepts, consistent with NO binding to each form in a simple reversible one-step mechanism. Fe(II) forms of MPO and LPO, but not EPO, displayed significantly lower affinity toward NO compared to Fe(III) forms, suggesting that heme reduction causes a dramatic change in the heme pocket electronic environment that alters the affinity and/or accessibility of heme iron toward NO. Optical absorbance spectra indicate that CO binds to the Fe(II) forms of both LPO and EPO, but not with MPO, and generates their respective low-spin six-coordinate complexes. Kinetic analyses indicate that the binding of CO to EPO is monophasic while CO binding to LPO is biphasic. Collectively, these results illustrate for the first time functional differences in the heme pocket environments of Fe(II) forms of EPO, LPO, and MPO toward binding of diatomic ligands. Our results suggest that, upon reduction, the heme pocket of MPO collapses, LPO adopts two spectroscopically and kinetically distinguishable forms (one partially open and the other relatively closed), and EPO remains open.

Nitric oxide modulates the catalytic activity of myeloperoxidase.

Myeloperoxidase (MPO), an abundant protein in neutrophils, monocytes, and subpopulations of tissue macrophages, is believed to play a critical role in host defenses and inflammatory tissue injury. To perform these functions, an array of diffusible radicals and reactive oxidant species may be formed through oxidation reactions catalyzed at the heme center of the enzyme. Myeloperoxidase and inducible nitric-oxide synthase are both stored in and secreted from the primary granules of activated leukocytes, and nitric oxide (nitrogen monoxide; NO) reacts with the iron center of hemeproteins at near diffusion-controlled rates. We now demonstrate that NO modulates the catalytic activity of MPO through distinct mechanisms. NO binds to both ferric (Fe(III), the catalytically active species) and ferrous (Fe(II)) forms of MPO, generating stable low-spin six-coordinate complexes, MPO-Fe(III).NO and MPO-Fe(II).NO, respectively. These nitrosyl complexes were spectrally distinguishable by their Soret absorbance peak and visible spectra. Stopped-flow kinetic analyses indicated that NO binds reversibly to both Fe(III) and Fe(II) forms of MPO through simple one-step mechanisms. The association rate constant for NO binding to MPO-Fe(III) was comparable to that observed with other hemoproteins whose activities are thought to be modulated by NO in vivo. In stark contrast, the association rate constant for NO binding to the reduced form of MPO, MPO-Fe(II), was over an order of magnitude slower. Similarly, a 2-fold decrease was observed in the NO dissociation rate constant of the reduced versus native form of MPO. The lower NO association and dissociation rates observed suggest a remarkable conformational change that alters the affinity and accessibility of NO to the distal heme pocket of the enzyme following heme reduction. Incubation of NO with the active species of MPO (Fe(III) form) influenced peroxidase catalytic activity by dual mechanisms. Low levels of NO enhanced peroxidase activity through an effect on the rate-limiting step in catalysis, reduction of Compound II to the ground-state Fe(III) form. In contrast, higher levels of NO inhibited MPO catalysis through formation of the nitrosyl complex MPO-Fe(III)-NO. NO interaction with MPO may thus serve as a novel mechanism for modulating peroxidase catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo.

Subunit dissociation and unfolding of macrophage NO synthase: relationship between enzyme structure, prosthetic group binding, and catalytic function.

Macrophage NO synthase is a homodimer of 130 kDa subunits. Each subunit contains an oxygenase domain that binds iron protoporphyrin IX (heme) and tetrahydrobiopterin (H4biopterin) and a reductase domain that binds FAD, FMN, and calmodulin (CaM) [Ghosh & Stuehr (1995) Biochemistry 34, 801-807]. We have studied the dissociation and unfolding reactions of dimeric iNOS in urea to learn how enzyme structure relates to catalysis and prosthetic group binding. The iNOS dimer dissociated between 0 and 2.5 M urea, and the subunits partially unfolded at 2.5 M urea and above. Dimer dissociation was accompanied by loss of NO synthesis activity and release of bound H4biopterin from the protein. However, the dissociated subunits maintained their cytochrome c and ferricyanide reductase activities and retained near stoichiometric quantities of bound heme. The subunit unfolding transition was accompanied by loss of reductase activities and partial loss of bound heme but retention of bound flavins and CaM. The heme iron in the dissociated subunits remained coordinated through axial cysteine thiolate ligation. Kinetic analysis of dimer dissociation showed that loss of NO synthesis correlated with a loss of heme Soret absorbance at 398 nm and an appearance of absorbance bands at 377 and 460 nm, which were attributed to DTT coordination to the sixth position of the heme iron to form a mixed bisthiolate complex. Subunits could reassociate into a dimer when incubated with L-arginine and H4biopterin. Dimer formation correlated with proportional recoveries of NO synthesis and heme Soret absorbance at 398 nm. Thus, dimeric iNOS undergoes separate dissociation and unfolding transitions in urea, and each transition is accompanied by a loss of a specific catalytic function.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of bacterial luciferase with aldehyde substrates and inhibitors.

Bacterial luciferase catalyzes the reaction of FMNH2, O2, and an aliphatic aldehyde to yield the carboxylic acid, FMN, water and blue-green light. The kinetics of the bacterial luciferase reaction were measured by stopped-flow spectrophotometry at pH 7 and 25 degrees C for the series of aldehydes from n-heptanal to n-undecanal. The rate of formation of the 4a-hydroperoxyflavin intermediate was dependent on the aldehyde concentration when mixtures of enzyme, FMNH2, and aldehyde were rapidly mixed with O2. At saturating aldehyde, the rate of formation of this intermediate was 100-fold slower than in the absence of aldehyde, demonstrating that an enzyme.FMNH2.aldehyde complex can be formed. Numerical simulation of the time courses for these experiments supported the formation of this intermediate and its direct reaction with O2. The kinetics of the light emitting reaction were dependent upon the chain length of the aldehyde substrate. Although the initial light intensity and the light emission decay rate were different for each aldehyde, the quantum yield for the reaction was independent of the aldehyde used. Luciferase was inhibited by high levels of the aldehyde substrate when the enzyme was assayed by mixing FMNH2 with an aerobic mixture of enzyme and aldehyde. The extent of inhibition was dependent on the particular aldehyde used, and the binding affinity of the aldehyde for the free enzyme increased in parallel with the aldehyde chain length. The kinetics of the formation and decay of the various intermediates were also studied in the presence of n-alkyladehyde analogs. These compounds decreased the rate of formation of the 4a-hydroperoxyflavin intermediate in much the same way as the aldehyde substrate, presumably by the formation of the enzyme.FMNH2.analog ternary complex.

Calmodulin controls neuronal nitric-oxide synthase by a dual mechanism. Activation of intra- and interdomain electron transfer.

In neuronal nitric-oxide synthase (NOS), electron transfer proceeds across domains in a linear sequence from NADPH to flavins to heme, with calmodulin (CaM) triggering the interdomain electron transfer to the heme (Abu-Soud, H. M., and Stuehr, D. J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10769-10772). Here, we utilized a neuronal NOS devoid of its bound heme and tetrahydrobiopterin (apo-NOS) to examine whether interdomain electron transfer is responsible for CaM's activation of NO synthesis, substrate-independent NADPH oxidation, and cytochrome c and ferricyanide reduction. Of the four activities, two (cytochrome c and ferricyanide reduction) were similarly stimulated by CaM in apo-NOS when compared with native NOS, indicating that activation occurs by a mechanism not involving flavin-to-heme electron transfer. Further analysis showed that CaM increased the rate of electron transfer from NADPH into the flavin centers by a factor of 20, revealing a direct activation of the NOS reductase domain by CaM. In contrast, CaM's activation of NO synthesis and substrate-independent NADPH oxidation appeared to involve flavin-to-heme electron transfer because these reactions were not activated in apo-NOS and were blocked in native NOS by agents that prevent heme iron reduction. Thus, CaM activates neuronal NOS at two points in the electron transfer sequence: electron transfer into the flavins and interdomain electron transfer between the flavins and heme. Activation at each point is associated with an up-regulation of domain-specific catalytic functions. The dual regulation by CaM is unique and represents a new means by which electron transfer can be controlled in a metalloflavoprotein.

Electron transfer in the nitric-oxide synthases. Characterization of L-arginine analogs that block heme iron reduction.

Heme iron reduction in the nitric-oxide synthases (NOSs) requires calmodulin binding and is associated with increased NO synthesis and NADPH oxidation (Abu-Soud, H. M., and Stuehr, D. J. (1993) Proc. Natl. Acad. Sci., U. S. A. 90, 10769-10772). Here, we examined how L-arginine and the analogs N omega-methyl-L-arginine (NMA), N omega-nitro-L-arginine methyl ester (NAME), and d-(thioureido)-L-norvaline (thiocitrulline) affect electron flux through neuronal and macrophage NOS. L-Arginine and NMA increased or decreased NOS NADPH consumption depending on the isoform, while thiocitrulline and NAME decreased NADPH oxidation in both NOS by 73-86% relative to their ligand-free rates. Kinetic studies showed that thiocitrulline and NAME inhibited NOS NADPH consumption through binding within the substrate binding site. Thiocitrulline and NAME did not affect the NADPH-dependent reduction of NOS flavins nor NOS cytochrome c reduction, indicating that they blocked electron flux at a point beyond the flavins in the electron transfer sequence. Thiocitrulline and NAME inhibited both NADPH-dependent and dithionite-mediated heme iron reduction in the NOS isoforms relative to the substrate-free NOS, whereas L-arginine and NMA did not. Thus, L-arginine and NMA increase or decrease electron flux through the NOS by coupling NADPH oxidation to NO synthesis (L-arginine), or by occupying the substrate binding site with minimal catalytic coupling (NMA). In contrast, thiocitrulline and NAME decrease electron flux through both NOS isoforms by decreasing the reduction potential of the heme iron. Inhibition of heme iron reduction by substrate analogs is unusual and represents a new means to modulate electron flow through the NOS.

Heme coordination of NO in NO synthase.

A current question in nitric oxide (NO) biology is whether NO can act as a feedback inhibitor of NO synthase (NOS). We have approached this problem by examining the interaction of NO with neuronal NOS by optical absorption and resonance Raman scattering spectroscopies. Under an inert atmosphere NO coordinated to the heme iron in both the oxidized and reduced forms of NOS. The Soret and visible optical absorption transitions are detected at 436 and at 567 nm, respectively, in the Fe(2+)-NO heme complex and at 440 nm and at 549 and 580 nm, respectively, in the Fe(3+)-NO heme complex. In the resonance Raman spectrum of the ferrous complex the Fe-NO stretching mode is located at 549 cm-1 in the presence of L-arginine and at 536 cm-1 in the absence of L-arginine, whereas in the ferric enzyme the mode is located at 540 cm-1 (in the absence of L-arginine). The interaction between bound L-arginine and the NO indicates that L-arginine binds directly over the heme just as do the substrates in cytochrome P-450s. In the absence of L-arginine, NO readily oxidized the ferrous heme iron. The oxidation was prevented by the presence of bound L-arginine and enabled NOS to form a stable ferrous NO complex. Under oxygen-limited conditions, NO generated by neuronal NOS coordinated to its heme iron and formed a spectrally detectable ferrous-NO complex. Taken together, our results show that NO can bind to both ferric and ferrous NOS and may inhibit NO synthesis through its binding to the heme iron during catalysis.

Regulation of inducible nitric oxide synthase by self-generated NO.

A ferric heme-nitric oxide (NO) complex can build up in mouse inducible nitric oxide synthase (iNOS) during NO synthesis from L-arginine. We investigated its formation kinetics, effect on catalytic activity, dependence on solution NO concentration, and effect on enzyme oxygen response (apparent KmO2). Heme-NO complex formation was biphasic and was linked kinetically to an inhibition of electron flux and catalysis in iNOS. Experiments that utilized a superoxide generating system to scavenge NO showed that the magnitude of heme-NO complex formation directly depended on the NO concentration achieved in the reaction solution. However, a minor portion of heme-NO complex (20%) still formed during NO synthesis even when solution NO was completely scavenged. Formation of the intrinsic heme-NO complex, and the heme-NO complex related to buildup of solution NO, increased the apparent KmO2 of iNOS by 10- and 4-fold, respectively. Together, the data show heme-NO complex buildup in iNOS is due to both intrinsic NO binding and to equilibrium binding of solution NO, with the latter predominating when NO reaches high nanomolar to low micromolar concentrations. This behavior distinguishes iNOS from the other NOS isoforms and indicates a more complex regulation is possible for its activity and oxygen response in biologic settings.

The ferrous-dioxy complex of neuronal nitric oxide synthase. Divergent effects of L-arginine and tetrahydrobiopterin on its stability.

Nitric oxide synthases (NOS) are hemeproteins that catalyze oxidation of L-arginine to nitric oxide (NO) and citrulline. The NOS heme iron is expected to participate in oxygen activation during catalysis, but its interactions with O2 are not characterized. We utilized the heme-containing oxygenase domain of neuronal NOS (nNOSoxy) and stopped-flow methods to study formation and autooxidative decomposition of the nNOSoxy oxygenated complex at 10 degrees C. Mixing ferrous nNOSoxy with air-saturated buffer generated a transient species with absorption maxima at 427 and approximately 560 nm. This species decayed within 1 s to form ferric nNOSoxy. Its formation was first order with respect to O2, monophasic, and gave rate constants for kon = 9 x 10(5) M-1 s-1 and koff = 108 s-1 for an L-arginine- and tetrahydrobiopterin (H4B)-saturated nNOSoxy. Omission of L-arginine and/or H4B did not greatly effect O2 binding and dissociation rates. Decomposition of the oxygenated intermediate was independent of O2 concentration and was either biphasic or monophasic depending on sample conditions. L-Arginine stabilized the oxygenated intermediate (decay rate = 0.14 s-1), while H4B accelerated its decay by a factor of 70 irrespective of L-arginine. The spectral and kinetic properties of the intermediate identify it as the FeIIO2 complex of nNOSoxy. Destabilization of a metallo-oxy species by H4B is unprecedented and may be important regarding the role of this cofactor in NO synthesis.