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J. Biol. Chem., Vol. 276, Issue 18, 14533-14536, May 4, 2001

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MINIREVIEW
Oxygen Reduction by Nitric-oxide Synthases^*

Dennis Stuehr, Sovitj Pou^§, and Gerald M. Rosen^§¶

From the  Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, ^§ Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201, and ^¶ Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201

	INTRODUCTION

TOP INTRODUCTION Relevant NOS Enzymology NOS-catalyzed Production of O2 Electron Acceptors and Drugs... Superoxide from NOS-containing... Oxygen Reduction Catalyzed by... Autooxidation of Bound... NOS Electron Transfer Kinetics New Role for H4B... Electron Flux from NOS... Summary REFERENCES

Nitric-oxide synthases (NOS,¹ EC 1.14.13.39) are hemeproteins that catalyze oxidation of L-arginine to NO^· and L-citrulline. Three main isozymes exist in mammals that are regulated by distinct genes (1-6): a constitutive neuronal NOS (nNOS or NOS I), (7, 8), an endotoxin- and cytokine-inducible NOS (iNOS or NOS II) (9, 10), and a constitutive endothelial NOS (eNOS or NOS III) (11). Although NOS have some unique features that distinguish them from other hemeprotein monooxygenases such as cytochrome P-450s, there are, nevertheless, a number of similarities that allow comparisons to be made (12). One such finding is that both enzymes inadvertently secrete O₂ (13, 14). Indeed, NOSs contain four redox active prosthetic groups (FAD, FMN, iron protoporphyrin IX (heme), and (6R)-tetrahydrobiopterin (H₄B)) that could conceivably pass electrons to O₂. Understanding the extent to which this occurs independent of NO^· synthesis is important from a mechanistic standpoint. In particular, how does the enzyme control O₂ activation? From a biologic perspective, NO^· and O₂ initiate different cell signaling pathways (15, 16). This is further complicated by the fact that NO^·and O₂ combine to form peroxynitrite (17), which has physiological activities that differ greatly from those of the parent free radicals (18). In this review we examine the electron transport chain of NOS with special emphasis on O₂ production and interpret these findings with a view toward NOS structure-function and the kinetics of the electron transfer reactions.

	Relevant NOS Enzymology

TOP INTRODUCTION Relevant NOS Enzymology NOS-catalyzed Production of O2 Electron Acceptors and Drugs... Superoxide from NOS-containing... Oxygen Reduction Catalyzed by... Autooxidation of Bound... NOS Electron Transfer Kinetics New Role for H4B... Electron Flux from NOS... Summary REFERENCES

In NOS, electrons from NADPH are used to reduce and activate O₂ at the heme, with H₂O generated as a co-product. Synthesis of NO^· requires the enzyme to cycle twice (Fig. 1). In the first step, NOS consumes 1 mol of NADPH to hydroxylate L-arginine to N-hydroxyl-L-arginine, which is an enzyme-bound intermediate. Thereafter, NOS consumes 0.5 mol of NADPH to oxidize N-hydroxyl-L-arginine to citrulline and NO^· (19). Given that NOS must bind and activate O₂ twice to generate NO^· from L-arginine, the enzyme would need to carefully control the quantity and tempo of electron transfer to minimize uncoupled O₂ reduction during the reaction.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Stepwise NO synthesis by NOS. Symbols (, *) trace the sources of nitrogen and oxygen in the products.

NOS subunits are comprised of two domains connected by a central Ca²⁺/calmodulin-binding region (20). Fig. 2 illustrates the electron transport pathway as proposed for a NOS II dimer. To accomplish O₂ activation, electrons from NADPH transfer into a reductase domain that contains FAD and FMN and then pass from the FMN to an oxygenase domain that contains bound heme, H₄B, and substrate. The reduced heme can then bind and activate O₂ for NO^· synthesis. A similar electron transfer sequence has been shown for cytochrome P-450 (13, 21), although in that case the flavoprotein and hemeprotein components are typically separate monomeric entities.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Electron transfer pathway in a NOS II dimer. CAM, calmodulin. From Ref. 81.

The reductase domain of NOS actually catalyzes three separate electron transfer reactions: (a) NADPH reduction of bound FAD via hydride transfer, (b) subsequent distribution of single electrons between FAD and FMN (disproportionation), and (c) electron transfer from reduced FMN to either the NOS heme or to an artificial acceptor like ferricytochrome c. Calmodulin binding to NOS speeds electron transfer at points a, b, and c in the reductase domains of NOS I and NOS III (22-25). As such the rates become equivalent to those seen in related flavoproteins like sulfite oxidase and cytochrome P-450 reductase (26). Calmodulin may function by relieving a repression brought on by two unique negative control elements that are present in the NOS reductase domains (27-32). One control element is located in the FMN module and the other at the C terminus of the reductase domain. Calmodulin may cause the control element in the FMN module to interact with the C-terminal element and relieve its repression of electron transfer from NADPH to FAD. Because NOS II contains tightly bound calmodulin and is missing the FMN module control element, its flavoprotein domain is never repressed regarding the three electron transfer reactions noted above (26, 27). Each flavin in NOS can shuttle between its fully oxidized, semiquinone, or two-electron reduced form (e.g. FAD, FADH^·, and FADH₂). However, thermodynamic measurements on the NOS I reductase domain indicate that only fully reduced FMN (e.g. FMNH₂) is capable of reducing the ferric heme of the enzyme (33).

Oxygen activation by NOS heme appears to occur in steps and involves transfer of two electrons singly to heme (Fig. 3). The first electron transfer to heme can only come from the reductase domain (i.e. H₄B and L-arginine are not donors) (34). However, the second electron provided to heme can derive either from the reductase domain, H₄B, or N-hydroxyl-L-arginine (34-38). Recent reports suggest that H₄B is kinetically preferred over the reductase domain as a source of the second electron (34-36) and may discount electron donation by N-hydroxyl-L-arginine, although its potential to donate cannot be ruled out based on theoretical studies (39).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Model for oxygen reduction by NOS. Electron transfer from the reductase domain enables NOS ferric heme to bind O2 and form a ferrous-dioxy species. This species may receive a second electron from H4B or the reductase domain, and this allows formation of putative heme-peroxy and heme-oxo species (in brackets) that are thought to catalyze Arg hydroxylation. Alternatively, the ferrous dioxy species can decay to generate O2 (bold), and the heme-peroxo species may decay to generate H2O2 (bold). Rates of various electron transfer and decay reactions are in parentheses and were obtained from the literature. See "Relevant NOS Enzymology" for details.

Importantly, NOS heme-oxy intermediates are unstable and will either release O₂ or H₂O₂ if electrons are not delivered to the heme at a sufficient rate or if substrate is not present (Fig. 3). Thus, a central challenge for all NOS is to arrange that electron transfer events be maximally coupled for NO^· synthesis and in this way minimize uncoupled reduction of O₂.

	NOS-catalyzed Production of O2

TOP INTRODUCTION Relevant NOS Enzymology NOS-catalyzed Production of O2 Electron Acceptors and Drugs... Superoxide from NOS-containing... Oxygen Reduction Catalyzed by... Autooxidation of Bound... NOS Electron Transfer Kinetics New Role for H4B... Electron Flux from NOS... Summary REFERENCES

NOS has been known to generate O₂ and H₂O₂ (at least through dismutation) since the early 1990s (14, 40-45). This occurs during NADPH oxidation in the absence of L-arginine and is inhibited by the addition of L-arginine in a concentration-dependent manner (44). Superoxide was directly identified by spin trapping/EPR spectroscopy, whereas H₂O₂ was presumed to derive from O₂ dismutation. However, under these experimental conditions, it is important to note that spin traps can only qualitatively estimate O₂ production because of a relatively slow rate of reaction (k  90 M¹ s¹, physiological pH, Refs. 46 and 47) compared with self-dismutation (k = 3.0 × 10⁵ M¹ s¹, pH 7.4, Ref. 48) plus an inherent instability of the corresponding spin trapped adducts.

	Electron Acceptors and Drugs That Boost NOS Release of O2

TOP INTRODUCTION Relevant NOS Enzymology NOS-catalyzed Production of O2 Electron Acceptors and Drugs... Superoxide from NOS-containing... Oxygen Reduction Catalyzed by... Autooxidation of Bound... NOS Electron Transfer Kinetics New Role for H4B... Electron Flux from NOS... Summary REFERENCES

One of the first studies that tested O₂ formation by the reductase domain found that the chemotherapeutic drug, adriamycin, enhanced NOS III production of O₂ in the absence of L-arginine (49). More recently, other foreign compounds including lucigenin, nitroblue tetrazolium, 2,6-dichlorophenolindophenol, and quinones (44, 50-53) have been found to uncouple the electron transport of NOS. In some cases this has led to enhanced production of O₂ even though L-arginine is present. One such xenobiotic is the herbicide paraquat (54). Under anaerobic conditions, NOS will reduce paraquat to a paraquat free radical. In the presence of O₂, O₂ is generated (55). It has been proposed that paraquat-induced toxicity may be mediated through a mechanism that uncouples the electron transport chain of NOS (53).

	Superoxide from NOS-containing Cells

TOP INTRODUCTION Relevant NOS Enzymology NOS-catalyzed Production of O2 Electron Acceptors and Drugs... Superoxide from NOS-containing... Oxygen Reduction Catalyzed by... Autooxidation of Bound... NOS Electron Transfer Kinetics New Role for H4B... Electron Flux from NOS... Summary REFERENCES

The versatility of spin trapping allows free radicals to be detected in cell suspensions (56). Not surprisingly, NOS-dependent cellular secretion of NO^· and O₂, as monitored by the spin traps ferro-N-methyl-D-glucamine dithiocarbamate and 5,5-dimethyl-1-pyrroline-N-oxide, was identified from control and L-arginine-depleted cells (57-59). NO^· and O₂ were detected outside the cells, consistent with their having a lifetime sufficiently long to diffuse from the intracellular site where NOS was localized. NOS-secreted O₂ from neurons occurred in response to glutamate receptor stimulation (59), which elevates intracellular Ca²⁺ and activates NOS I via calmodulin binding. Under circumstances of prolonged Ca²⁺ influx, NOS I began to generate O₂ after 10-15 min, consistent with a need to deplete intracellular stores of L-arginine. Superoxide production by NOS II occurred in L-arginine-depleted macrophages after enzyme expression was induced by inflammatory cytokines (58) and was associated with protein tyrosine nitration, suggesting NOS-derived O₂ may have combined with NO^· to generate ONOO. These findings complement results with pure enzymes and show how a low intracellular L-arginine concentration can predispose NOS to generate O₂ by catalyzing uncoupled O₂ reduction at its heme.

	Oxygen Reduction Catalyzed by NOS Prosthetic Groups

TOP INTRODUCTION Relevant NOS Enzymology NOS-catalyzed Production of O2 Electron Acceptors and Drugs... Superoxide from NOS-containing... Oxygen Reduction Catalyzed by... Autooxidation of Bound... NOS Electron Transfer Kinetics New Role for H4B... Electron Flux from NOS... Summary REFERENCES

How well NOS prosthetic groups transfer electrons to O₂ depends on the surrounding protein structure and on the thermodynamics and kinetics of the electron transfer reactions. Such biochemical data are available and can help explain NOS O₂ production under various circumstances, as well as gauge its relative importance.

	Autooxidation of Bound Prosthetic Groups

TOP INTRODUCTION Relevant NOS Enzymology NOS-catalyzed Production of O2 Electron Acceptors and Drugs... Superoxide from NOS-containing... Oxygen Reduction Catalyzed by... Autooxidation of Bound... NOS Electron Transfer Kinetics New Role for H4B... Electron Flux from NOS... Summary REFERENCES

NOS reductase domains belong to a class of flavoprotein dehydrogenases that are sterically constrained against binding O₂ to their reduced flavins (60). This severely limits two-electron reduction of O₂ by the flavins to generate H₂O₂, but typically it still allows one-electron transfer to generate O₂. How well NOS proteins prevent autooxidation of their reduced flavins can be appreciated by comparing autooxidation rates of free and NOS-bound flavin. In NOS, flavin autooxidation rates range from 0.01 to 0.03 s¹ (Table I), which is about 1000 times slower than free FADH₂ in aerated solution (61). One of the two negative control elements present in the NOS reductase domain appears to help protect the flavins from autooxidation (31).

View this table: [in this window] [in a new window]   Table I Kinetic constants related to O2 reduction in NOS isoforms Rates are listed in units of s1 and refer to enzyme-bound cofactors. Rates were determined at 10 °C unless specified otherwise. NA, not available.

A similar situation exists for H₄B in NOS. Free H₄B oxidizes in solution to generate O₂ but at a much slower rate than reduced flavins (62). In NOS H₄B binds next to the heme and near the dimer interface (63, 64) and is stabilized to such an extent that its resistance to autooxidation is practically absolute. In contrast, NOS heme is bound at the bottom of a solvent-exposed substrate binding channel (63, 64) and can bind O₂ quite rapidly whether L-arginine is bound or not (25, 65) (Table I). The NOS heme can reduce O₂ by one or two electrons (Fig. 3). Interestingly, oxidative decay of the ferrous heme-O₂ species occurs much faster in NOS than in related heme-thiolate proteins like cytochrome P-450 (37, 65, 66). These decay rates differ somewhat between the NOS isoforms (Table I) and appear to be faster in full-length NOS compared with the oxygenase domain. Oxidative decay of NOS ferrous heme-O₂ species is increased when H₄B is bound (35, 37, 62, 63), and the effect is not influenced by substrate. The basis may relate to a redox function for H₄B (see section below). When one compares autooxidation rates of bound flavins and ferrous heme in NOS (Table I), the heme is far faster and thus would be predicted to be the most prominent source of reduced O₂ species in calmodulin-bound NOS when substrate is not present.

	NOS Electron Transfer Kinetics

TOP INTRODUCTION Relevant NOS Enzymology NOS-catalyzed Production of O2 Electron Acceptors and Drugs... Superoxide from NOS-containing... Oxygen Reduction Catalyzed by... Autooxidation of Bound... NOS Electron Transfer Kinetics New Role for H4B... Electron Flux from NOS... Summary REFERENCES

To understand how electron transfer into NOS flavins and heme relates to their capacity to reduce O₂, Table I summarizes available kinetic data on flavin and heme reduction rates for the various NOS isozymes. Flavin reduction appears as a biphasic process that involves both FAD reduction by NADPH and electron disproportionation into FMN (23, 24, 27, 32, 34). These steps are relatively fast and are markedly enhanced upon calmodulin binding. Although differences exist between the three NOS isozymes, their rates of flavin reduction far exceed rates of flavin autooxidation in all cases. Interestingly, calmodulin binding has a negligible effect on flavin autooxidation, despite calmodulin increasing the rate of flavin reduction and causing conformational change in the FMN module of the reductase domain (32). These data establish the slow step in NOS flavin autooxidation to be their electron transfer to O₂ and underscore the role of protein in minimizing this process.

Flavins in all three NOS isozymes transfer electrons more slowly to their hemes than they receive electrons from NADPH (Table I). However, heme reduction is still faster than flavin autooxidation. When H₄B is bound, the flavins reduce ferric heme more slowly than autooxidation of the ferrous heme-O₂ species (Table I and Fig. 3). This means that heme reduction should limit the rate of reduced oxygen species production by substrate-free, calmodulin-bound NOS.

	New Role for H4B in O2 Activation

TOP INTRODUCTION Relevant NOS Enzymology NOS-catalyzed Production of O2 Electron Acceptors and Drugs... Superoxide from NOS-containing... Oxygen Reduction Catalyzed by... Autooxidation of Bound... NOS Electron Transfer Kinetics New Role for H4B... Electron Flux from NOS... Summary REFERENCES

Recent evidence suggests that H₄B may provide an electron to the ferrous heme-O₂ species during stepwise O₂ activation (35-37) (Fig. 3). The rate of this reaction in NOS II (Table I) is faster than flavoprotein reduction of the ferric heme. Thus, by quickly donating an electron H₄B may minimize oxidative decay of the ferrous heme-O₂ species, which is a process that competes kinetically and generates O₂ at the expense of NO^· synthesis (34). H₄B appears to function identically in substrate-free NOS II (36). In that case, quick reduction of the ferrous heme-O₂ species by H₄B may promote direct H₂O₂ production by the NOS heme at the expense of O₂ release (Fig. 3). This mechanism could explain why H₄B lowers NOS O₂ formation while it promotes NADPH oxidation. Indeed, supplementation of substrate-free NOS I with as little as 10 µM H₄B inhibits its production of O₂ (42, 44).

	Electron Flux from NOS to O2

TOP INTRODUCTION Relevant NOS Enzymology NOS-catalyzed Production of O2 Electron Acceptors and Drugs... Superoxide from NOS-containing... Oxygen Reduction Catalyzed by... Autooxidation of Bound... NOS Electron Transfer Kinetics New Role for H4B... Electron Flux from NOS... Summary REFERENCES

Rates of O₂ reduction by substrate-free NOS relate directly to their rates of NADPH oxidation when these are measured in the absence of any other electron acceptor besides O₂. The relative contributions of NOS flavins and heme to the O₂ reduction rate, as well as the effect of calmodulin binding, can be discerned by comparing NADPH oxidation by isolated reductase domains, heme-free NOS, or NOS whose heme reduction is inhibited with sodium cyanide, imidazole, or N-nitro-L-arginine.

Table II shows that rates of NADPH oxidation are slow when only the NOS flavins are allowed to transfer electrons to O₂, even with calmodulin bound. Rates in NOS I and II are substantially less than 0.1 s¹, but in NOS III those rates are reported to be 0.1 s¹. Although the reason for this difference is unclear, it might be related to FAD and FMN molecules in solution catalyzing electron transfer from NOS to O₂ (42). Table II also shows that NADPH oxidation rates are enhanced when NOS heme is allowed to receive electrons and catalyze O₂ reduction. NADPH oxidation rates in substrate-free NOS isoforms vary according to their rates of heme reduction (NOS I > NOS II > NOS III), consistent with this step being rate-limiting.

In general, these steady-state data are consistent with slow autooxidation rates observed for the NOS flavoproteins (Table I) and confirm that NOS flavins are relatively poor O₂ generators. However, these data also suggest a potentially important distinction between NOS III and the other two NOS isozymes regarding O₂ generation during NO^· synthesis. Namely, heme reduction in NOS III is so slow that flavin-mediated O₂ reduction may be a significant alternative. Spin trapping experiments appear to confirm this hypothesis (42, 43). The amount of O₂ produced during NO^· synthesis would directly depend on O₂ concentration, because it will effect the rate of flavin autooxidation but not the rate of heme reduction. In contrast, heme reduction in NOS I and II is so much faster than flavin autooxidation that one expects little O₂ production resulting from flavin autooxidation during their NO^· synthesis.

Although flavin autooxidation in NOS is expected to generate O₂, the heme can conceivably generate both O₂ and H₂O₂ in the absence of substrate. Product partitioning in this circumstance depends on the relative rates of ferrous heme-O₂ oxidative decay versus its reduction to a heme-peroxo species (Fig. 3). As noted previously, H₄B may tip the scales toward reduction by providing an electron more quickly to the ferrous heme-O₂ species. If this holds true, then H₄B may enable NOS to generate more H₂O₂ than O₂ during NADPH oxidation in the absence of substrate. Careful testing of the product ratio is challenging but ultimately will be important for understanding the impact of the NOS-generated reactive O₂ species.

	Summary

TOP INTRODUCTION Relevant NOS Enzymology NOS-catalyzed Production of O2 Electron Acceptors and Drugs... Superoxide from NOS-containing... Oxygen Reduction Catalyzed by... Autooxidation of Bound... NOS Electron Transfer Kinetics New Role for H4B... Electron Flux from NOS... Summary REFERENCES

Four conclusions derive from the studies exploring NOS-generated O₂ and the kinetic data outlined here. 1) When sufficient L-arginine and H₄B are present, NOS dimers secrete small amounts of O₂ or H₂O₂ and instead couple their heme and O₂ reduction to NO^· synthesis. 2) Significant O₂ production may occur when concentrations of H₄B (2 µM) or L-arginine (100 µM) fall below levels required to saturate the enzyme. In these circumstances, O₂ forms by heme-catalyzed O₂ reduction. 3) NOS reductase domain flavins are protected from autooxidation and do not secrete large amounts of O₂ unless certain redox-active xenobiotics are present. These cause O₂ production by catalyzing electron transfer from the NOS reductase domain to O₂. One recent example suggests that NOS-derived O₂ participates in tissue injury associated with the xenobiotic (54). 4) NOS may generate both NO^· and O₂ when concentrations of L-arginine or H₄B are low (41-45, 51, 59). When the steady-state flux of O₂ was high there was evidence for ONOO formation (57, 58). Indeed, certain pathologic states might promote formation of ONOO such as ischemia/reperfusion injury (67). Formation of HO^·, either through metal ion-catalyzed H₂O₂ decomposition (68) or from decomposition of ONOO (69-71), at sensitive cellular sites may also contribute to cytotoxicity. Finally, it is worth noting that sequential formation of NO^· and O₂ can result in differing cell signaling pathways (15, 16), few of which have been well defined. Therefore, under different conditions a variety of oxidants may derive from NOS that can impact cell function in ways that are significant.

	ACKNOWLEDGEMENT

We thank Dr. Koustubh Panda for assistance and discussions.

	FOOTNOTES

^* This minireview will be reprinted in the 2001 Minireview Compendium, which will be available in December, 2001. This work was supported in part by National Institutes of Health Grants RR-12257 and CA-69538 (to G. M. R.) and CA-53914 and GM-51491 (to D. S.).

To whom correspondence should be addressed: Dept. of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 725 W. Lombard St., Baltimore, MD 21201. Tel.: 410-706-0514; Fax: 410-706-8184; E-mail: grosen@umaryland.edu.

Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.R100011200

	ABBREVIATIONS

The abbreviations used are: NOS, nitric-oxide synthase(s); H₄B, (6R)-tetrahydrobiopterin.

	REFERENCES

TOP INTRODUCTION Relevant NOS Enzymology NOS-catalyzed Production of O2 Electron Acceptors and Drugs... Superoxide from NOS-containing... Oxygen Reduction Catalyzed by... Autooxidation of Bound... NOS Electron Transfer Kinetics New Role for H4B... Electron Flux from NOS... Summary REFERENCES

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