Characterization of the Magnitude and Mechanism of Aldehyde Oxidase-mediated Nitric Oxide Production from Nitrite*

Aldehyde oxidase (AO) is a cytosolic enzyme with an important role in drug and xenobiotic metabolism. Although AO has structural similarity to bacterial nitrite reductases, it is unknown whether AO-catalyzed nitrite reduction can be an important source of NO. The mechanism, magnitude, and quantitative importance of AO-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. The kinetics and magnitude of AO-dependent NO formation were characterized. In the presence of typical aldehyde substrates or NADH, AO reduced nitrite to NO. Kinetics of AO-catalyzed nitrite reduction followed Michaelis-Menten kinetics under anaerobic conditions. Under physiological conditions, nitrite levels are far below its measured Km value in the presence of either the flavin site electron donor NADH or molybdenum site aldehyde electron donors. Under aerobic conditions with the FAD site-binding substrate, NADH, AO-mediated NO production was largely maintained, although with aldehyde substrates oxygen-dependent inhibition was seen. Oxygen tension, substrate, and pH levels were important regulators of AO-catalyzed NO generation. From kinetic data, it was determined that during ischemia hepatic, pulmonary, or myocardial AO and nitrite levels were sufficient to result in NO generation comparable to or exceeding maximal production by constitutive NO synthases. Thus, AO-catalyzed nitrite reduction can be an important source of NO generation, and its NO production will be further increased by therapeutic administration of nitrite.

Nitric oxide (NO) 3 exerts a large number of important regulatory biological functions and also plays an important role in the pathogenesis of cellular injury (1)(2)(3)(4)(5). NO synthesis was first discovered in macrophages, endothelial cells, and neuronal cells (1, 6 -8). A group of enzymes were identified, NO synthases, which metabolize arginine to citrulline with the formation of NO (9,10). More recent studies have shown that in addition to NO generation from specific NO synthases, nitrite can be an important source of NO in biological tissues, especially under ischemic conditions (11)(12)(13)(14)(15)(16). However, questions remain regarding the precise mechanisms involved in this nitrite reduction.
Aldehyde oxidase (AO) (aldehyde:oxygen oxidoreductase; EC 1.2.3.1) is a cytosolic enzyme that plays an important role in the biotransformation of drugs and xenobiotics (17). AO belongs to the family of molybdenum-containing proteins with two iron-sulfur clusters, a flavin cofactor, and a molybdopterin cofactor (18,19). The similar molybdenum-containing enzyme xanthine oxidoreductase (XOR) has been shown previously to be a highly effective nitrite/nitrate reductase playing an important role in catalyzing NO generation from nitrite in mammalian tissues, especially under acidic conditions (14, 16, 20 -26).
AO is present in highest levels in the liver but is also broadly distributed in other tissues, such as lung, blood vessels, heart, and kidney (27)(28)(29)(30)(31). The amino acid sequence of AO and XOR are remarkably similar, with ϳ86% homology, and there is structural similarity of AO, XOR, and bacterial molybdenum nitrite reductase. Our recent studies have shown that AO can also function as a nitrite reductase catalyzing nitrite reduction to NO (21). However, questions remain concerning the mechanism, substrate specificity, magnitude, and quantitative importance of AO-mediated NO generation in biological systems.
Although AO and XOR have similar structure and amino acid sequences, their substrate specificity and inhibitor susceptibility are different (28). Both XOR and AO exhibit broad specificity, accepting a variety of reducing substrates, including purine, pteridine, aldehyde, and NADH (32). But AO catalyzes the oxidation of aldehydes and NADH more efficiently, with lower K m , whereas XOR has higher affinity for xanthine, hypoxanthine, pteridine, and purine (14,32,33).
Aldehydes and NADH are different site-specific electron donors for AO. Although aldehydes donate electrons to AO at the molybdenum site, NADH reduces AO at the FAD site (28). Because aldehydes and NADH are widely present in tissues, it is of critical importance to investigate the magnitude and kinetics of nitrite-dependent NO generation in the presence of these endogenous reducing substrates. This will enable characteriza-tion of the mechanism and pathophysiological importance of AO-mediated nitrite reduction.
To characterize AO-catalyzed NO production and its quantitative importance in biological systems, EPR spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. NO formation was shown to occur due to nitrite reduction at the molybdenum site, with either NADH or aldehydes serving as reducing substrates. The kinetic parameters for AO-mediated nitrite reduction were determined, enabling prediction of the magnitude of NO formation and delineation of the quantitative importance of this process in biological systems.
Purification of AO-Rat liver AO was purified as we recently reported (35). AO activity was determined by adding a suitable volume of enzyme solution to 1 ml of 50 mM potassium phosphate buffer, pH 7.8, containing 25 M DMAC as reducing substrate at 30°C and monitoring the decrease in absorbance at 398 nm. DMAC oxidations and absorbance changes were converted to units of enzyme activity (IU) using an extinction coefficient ⑀ ϭ 30.5 mM Ϫ1 cm Ϫ1 . One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 mol of DMAC/min at 30°C (35). The activity of freshly isolated AO was ϳ1.8 units/mg but declined over time. The purified AO was stored under liquid nitrogen until needed.
Rat Liver Preparation-Male Sprague-Dawley rats (250 -300 g) were heparinized with 500 units of heparin and anesthetized with intraperitoneal pentobarbital at a dose of 30 -35 mg/kg. The livers were excised and washed with HBSS (4°C) to remove any residual blood and then minced into small pieces. Minced tissues (ϳ0.15 g/ml in HBSS) were homogenized, and the protein concentration was determined by a modification of the Lowry method using the Sigma protein concentration assay kit.
EPR Spectroscopy-EPR measurements were performed using a Bruker EMX spectrometer with HS resonator operating at X-band. Measurements were performed at ambient temperature with a modulation frequency of 100 kHz, modulation amplitude of 2.5 G, microwave power of 20 milliwatt. NO formation was measured by spin trapping using the ferrous iron complex of MGD. Solid ferrous ammonium sulfate and MGD (molar ratio, 1:5) were added to the deoxygenated (argonpurged) PBS buffer with a final concentration of 0.1 mM in iron.
Quantitation of NO formation and trapping were performed by double integration of the observed EPR signal with comparison with that from a similar aqueous NO-Fe-MGD standard (24).
Chemiluminescence Measurements-The rate of NO production was measured using a Sievers 270B NO analyzer interfaced through a DT2821 A to D board to a personal computer. In the analyzer, NO is reacted with ozone-forming excited-state NO 2 , which emits light. Mixing of reagents and purging of NO from the reaction mixture were done at controlled temperature in a glass-purging vessel equipped with heating jacket and pressure monitoring device, which allowed maintaining atmospheric pressure inside the purging vessel within limits of 1 mm Hg by keeping water levels at the same height via adjustment of in and out gas flow valves as described by our previous investigations (21,24). The release of NO was quantified by analysis of the digitally recorded signal from the photomultiplier tube using specially designed data acquisition and analysis software developed in our laboratory. After an initial 30-s equilibration of the flow from the purging vessel to the detector, the signal provides the rate of NO formation over time (36). Calibration of the magnitude of NO production was determined from the integral of the signal over time compared with that from nitrite concentration standards (23,24,37).
Immunoassay of Guanylyl Cyclase Activity-Activation of soluble guanylyl cyclase (sGC) was measured by enzyme-linked immunoassay. After incubation of liver homogenates (10 mg/ml protein) or AO (0.01 mg/ml), NADH or aldehyde with 10 M nitrite, and with 1 ml of reaction buffer (10 ng sGC, 5 mM EDTA, 2 mM MgCl 2 , and 1 mM GTP in 1 ml PBS) at 37°C under anaerobic conditions, the protein in the samples was removed by a Millipore filter (centrifuged 2000 ϫ g, 10 min, at 4°C). The measurements of cGMP in the solution were performed by immunoassay using direct cGMP assay kit according to the manufacturer's product protocol. The standard curve was obtained with known amounts of cGMP.
Statistical Analysis-Values are expressed as the mean of at least three repeated measurements and reported Ϯ S.D., unless noted otherwise. Statistical significance of difference was evaluated by Student's t test. A p value of less than 0.05 was considered to indicate statistical significance.

AO-mediated Nitrite Reduction and NO Generation-To
investigate AO-mediated NO generation from nitrite, studies were performed using a chemiluminescence NO analyzer. NO was purged from the solution by argon and then reacted with ozone in the analyzer to form excited-state NO 2 , which emits light. This method provides direct measurement of the rate of NO generation as a function of time. Prior to the addition of AO, no detectable NO generation was seen from nitrite (1 mM) in the presence of DMAC (50 M) or NADH (100 M) (Fig. 1, A, trace b, and B, trace b). However, after addition of AO (0.01 mg/ml), prominent NO generation was triggered ( Fig. 1, A, trace a, and B, trace a).
Pathophysiological Levels of Reducing Substrates and Nitrite in AO-mediated NO Generation-AO exhibits broad specificity, accepting a variety of reducing substrates. AO has a high affinity (low K m ) for aldehydes and NADH, which are found in all living cells (32). To further quantitate the rates of NO generation from nitrite in the presence of physiological levels of these typical AO-reducing substrates, EPR spectroscopy was applied to directly measure NO generation under anaerobic conditions. 15 NO generated from [ 15 N]nitrite is paramagnetic and binds with high affinity to the water-soluble spin trap Fe 2ϩ -MGD forming the mononitrosyl iron complex that exhibits a characteristic doublet 15 NO-Fe 2ϩ -MGD spectrum, rather than the triplet observed with natural abundance 14 NO, enabling direct and selective detection of nitrite-derived NO formation. From the intensity of the observed spectrum, quantitative measurement of NO generation can be performed (12,27,38).
In the absence of nitrite, with mixture of AO (0.04 mg/ml) and its reducing substrate DMAC or NADH, no signal was observed ( Fig.  2A). With nitrite (100 M) and DMAC or NADH also no signal was seen (Fig. 2B). However, upon addition of AO, a marked 15  Kinetics of Nitrite-dependent NO Generation-To further characterize the mechanism and magnitude of AO-mediated NO formation, kinetic studies of the effects of nitrite concentration on the magnitude of NO generation were performed. The rate of NO formation derived from nitrite reduction was measured under anaerobic conditions using the NO analyzer. Following addition of nitrite (0.01-10 mM), prominent generation of NO was detected from AO with 100 M NADH (Fig. 4A) or 50 M DMAC (Fig. 4B) as electron donor. Typical Michaelis-Menten kinetics were observed as a function of nitrite concentration, and the apparent K m and V max values are shown inside each curve. The concentration dependence for reducing substrates was also determined in the presence of a fixed nitrite concentration, 1 mM. Each of the typical reducing substrates NADH and DMAC acted as electron donors to support AOcatalyzed nitrite reduction. Again, typical Michaelis-Menten kinetics were observed (Fig. 4, C and D), and the apparent values of K m and V max were determined by fitting the data to the Michaelis-Menten equation. The values for each reducing substrate are shown inside each curve. From these data, it is possible to predict the magnitude of AO-catalyzed NO formation as a function of nitrite concentration in the presence of physiological levels of endogenous AO-reducing substrates and to further determine the quantitative importance of this mechanism  of NO generation in a given biological system where AO substrate levels are known.
Determination of the Mechanism and Reaction Site of Nitrite Reduction-The effects of site-specific inhibitors of AO were studied to investigate the reaction sites involved in the process of the AO-mediated nitrite reduction with different reducing substrates (Fig. 5). Raloxifene binds to the molybdenum site of AO. It was observed that raloxifene inhibited AO-mediated nitrite reduction regardless of the type of reducing substrate present (Fig. 5C). Near total inhibition of NO generation was seen in the presence of either DMAC or NADH. Because raloxifene inhibits substrate binding at the molybdenum site of the enzyme, this suggests that nitrite binds to the reduced molybdenum site. DPI, which acts at the FAD site, inhibited AO-mediated nitrite reduction only when NADH was used as the reducing substrate, and it did not inhibit NO generation when DMAC was used (Fig. 5B). This suggests that NADH donates electrons to FAD, and then electrons are transported back to reduce the molybdenum that in turn reduces nitrite to NO. With the aldehyde electron donor DMAC, both AO reduction by the aldehyde and oxidation by nitrite take place at the molybdenum site.
Effects of pH on Nitrite-dependent NO Generation-Under ischemic conditions, marked intracellular acidosis occurs, and pH values in tissues, such as the heart, can fall to levels of 6.0 or below (12). To assess NO generation from nitrite under different physiological or pathological conditions and to further characterize the mechanism of AO-catalyzed nitrite reduction, experiments were performed to measure the effect of pH on the magnitude of NO generation from nitrite. Measurements were performed with 0.01 mg/ml AO in the presence of 100 M nitrite. As shown in Table 1, it was observed that maximum AO-catalyzed NO generation occurs at pH 6.0. When the pH was decreased to 5.0 or increased above 8.0, a decrease in the rate of NO generation was observed.
Effect of Oxygen Tension on AOmediated NO Generation from Nitrite-To quantitatively describe the effect of oxygen and further investigate the mechanism of AO-mediated nitrite reduction, chemiluminescence measurements of the rate of NO production were performed using molybdenum site electron donor DMAC and flavin site electron donor NADH under different oxygen tensions. NO formation from AOcatalyzed nitrite reduction was measured with continuous purging with air, 10, 5, 2, or 1% oxygen, corresponding to  oxygen concentrations in solution at 37°C of 214, 102, 51, 20, and 10 M, respectively. DMAC provides electrons to AO at the molybdenum site, the same site of nitrite binding to the enzyme. For DAMC as reducing substrate, the rate of AO-mediated NO formation from 1 mM nitrite was decreased with the increase of oxygen, and competitive inhibition was observed (Fig. 6A). With the determined V max ϭ 13.5 nmol⅐s Ϫ1 ⅐units Ϫ1 and K m ϭ 3.3 mM as shown in Fig. 4B, the inhibition constant K i of oxygen on AO-mediated nitrite reduction was calculated to be 3.8 M, as obtained from fitting to Equation 1 (␥ 2 Ͼ 0.95), NADH reacts with AO at the FAD site of the enzyme. With NADH as electron donor and purging with air, 10, 5, 2, or 1% oxygen, the oxygen does show typical competitive inhibition kinetics. For 1 mM nitrite with AO (0.01 mg/ml), NADH (100 mM), and superoxide dismutase (500 units/ml) for reactions in argon, 1, 2, 5, 10, and 21% oxygen, the rates of NO generation were 2.61 Ϯ 0.34, 2.47 Ϯ 0.24, 2.13 Ϯ 0.17, 1.47 Ϯ 0.15, 1.27 Ϯ 0.11, and 1.13 Ϯ 0.09 nmol⅐s Ϫ1 ⅐units Ϫ1 , respectively (Fig. 6B). The rate of NO generation decreased with the increase of pO 2 when NADH was the reducing substrate; however, even in air, prominent NO production was still present.
Effect of Nitrite Reduction on sGC Activity -NO exerts a large number of important regulatory biological functions, including vascular smooth muscle relaxation, neuronal signal transduction, and inhibition of platelet aggregation. The principal receptor for NO is sGC, which catalyzes the conversion of GTP to the second messenger molecule cGMP. To determine the effect of nitrite reduction on sGC activation, enzyme-linked immunoassays were performed to measure cGMP formation. After incubation of nitrite (10 M) with NADH or DMAC in the presence or absence of AO in the reaction buffer (10 ng of sGC, 5 mM EDTA, 2 mM MgCl 2 , and 1 mM GTP in 1 ml of PBS) under anaerobic conditions for 10 min, measurements of the formation of the sGC product cGMP were performed. With 10 M nitrite and 100 M NADH or 50 M DMAC in the reaction buffer in the absence of AO, no significant cGMP formation was detected. In the presence of AO (0.01 mg/ml), significant sGC activation was triggered by nitrite (10 M) with either NADH (100 M) or DMAC (50 M) as electron donor (Fig. 7).
NO Generation and sGC Activation from Rat Liver-To further access the importance of AOmediated nitrite reduction in the processes of NO formation and sGC activation, additional measurements were performed in rat liver. To investigate the magnitude of AO-dependent NO generation and sGC activation in rat liver, chemiluminescence NO analyzer and immunoassays of cGMP formation were performed. The addition of nitrite triggered a large amount of NO generation (Fig.  8A) and markedly increased cGMP (Fig. 8B). With the AO inhibitor raloxifene, NO generation was decreased by ϳ53% (Fig. 8A), and cGMP formation was inhibited ϳ49% (Fig. 8B).

DISCUSSION
Numerous recent studies have shown that nitrite can be an important source rather than just a product of NO, particularly under conditions of tissue ischemia with limited oxygenation and resulting acidosis (12-14, 16, 22-25, 39, 40). However, the mechanism of nitrite reduction in biological systems has been unclear.
AO has a similar structure to bacterial nitrite reductase. Our recent study further indicates that AO is an effective nitrite reductase in mammalian tissues (21). But questions remained regarding the biological importance of this pathway of NO production, as well as the mechanism, magnitude, and substrate specificity of this process. Therefore, we performed the current series of studies to measure the magnitude and kinetics of NO formation and sGC activation that arise due to AO-mediated nitrite reduction. Data obtained using EPR or the chemiluminescence NO analyzer confirmed that AO is an effective nitrite reductase under anaerobic conditions. It was observed that both its FAD sitereducing substrate NADH and molybdenum site-reducing substrate DMAC could act as electron donors to support this AOmediated nitrite reduction (Figs. 1-4).
AO belongs to the family of molybdenum-containing proteins with two iron-sulfur clusters, a flavin cofactor, and a molybdopterin cofactor (18,19). Addition of the molybdenum site-specific inhibitor raloxifene blocked nitrite reduction and NO formation with either NADH or DMAC as reducing substrates. Whereas NADH-mediated NO generation was inhibited by the flavin modifier DPI, NO generation from DMAC was unaffected. These results suggested that AO-mediated nitrite reduction occurs at the molybdenum site. Whereas aldehydes directly reduce the molybdenum center, NADH initially reduces the flavin, which subsequently transfers electrons to the molybdenum as shown in Scheme 1.
From the studies performed, it is clear that AO can catalyze NO generation from nitrite under anaerobic conditions. The key questions are as follows. What is the magnitude of this process? Do the levels of NO produced have functional significance? To address these critical questions, a kinetic model can be constructed that enables prediction of the magnitude of AOcatalyzed NO formation and understanding the quantitative importance of this mechanism of NO generation in biological systems. With DMAC or NADH as reducing substrates, the rate of NO generation followed typical Michaelis-Menten kinetics (Fig. 3). Reactions 1-3 can define the steps in the reaction mechanism, where E ox is the fully oxidized enzyme; E red is the two-electron reduced enzyme, and EЈ red is the 1-electron reduced enzyme. S refers to the reducing substrates of AO such as aldehyde and NADH, and P is the corresponding product. It should be noted  Aldehyde Oxidase-mediated NO Generation DECEMBER From Reactions 1-3 and Equation 2, the rate of NO generation can be derived, and this can be expressed in the form of the Michaelis-Menten Equation 3, where terms are defined as shown in Equations 4 and 5, It was observed that over a broad range of nitrite concentrations, Equation 3 provided a good fit to the experimental data measuring the rate of NO generation from AO in the presence of molybdenum site-binding reducing substrates DMAC or flavin site electron donor NADH (Fig. 4). Similar to XOR, the K m of nitrite for AO is ϳ3 mM, which far exceeds normal cellular levels, which are ϳ10 M (12). Therefore, AO-mediated NO generation will increase linearly with tissue nitrite concentration, and the rates can be estimated by the kinetic data shown in Fig. 4.
AO plays an important role in the biotransformation of drugs and xenobiotics (17). Some of the AO substrates, including the toxic metabolite of ethanol, acetaldehyde, are of toxicological or pharmacological importance. The results of this study indicate that both NADH and aldehydes such as DMAC are effective electron donors for AO-mediated nitrite reduction under anaerobic conditions. Under aerobic conditions, oxygen can bind to the flavin site of AO, accept an electron and produce superoxide as shown in Reactions 4 and 5, With molybdenum site electron donor DMAC as reducing substrate, AO-mediated NO generation is greatly inhibited by oxygen. Oxygen acts as a competitive inhibitor (K i ϳ 3.8 M) of AO-mediated NO production (Fig. 6A). With FAD site electron donor NADH as reducing substrate, nitrite reduction can occur at the molybdenum site of the enzyme with or without the binding of NADH at the FAD site. Without the binding of NADH with the FAD site free, both oxygen and nitrite can accept electrons from reduced AO, and oxygen acts as a competitive inhibitor for AO-mediated nitrite reduction. However, when the FAD site of AO is occupied by the binding of NADH, oxygen reduction is blocked. Thus, under aerobic conditions, AO-mediated NO formation is maintained at much higher levels with the FAD site-binding substrate, NADH, as reducing substrate (Fig. 6B). Thus, it would be expected that NADH would be the major electron donor for AO-mediated nitrite reduction under aerobic conditions. AO has a high affinity for NADH with a K m value of ϳ24 M that is well below the tissue levels reported in normal and disease conditions (41). Therefore, this process could serve as an important pathway of nitrite reduction to produce NO in tissues under aerobic conditions. NADH is necessary for many biochemical reactions within the body and is found in every living cell. Under ischemic conditions, NAD ϩ is reduced, and NADH concentrations will be increased. In rat liver cytosol, the total amount of NADH was estimated to be ϳ270 M, and the total amount of NAD ϩ and NADH is ϳ1 mM (41). In myocardial tissue, intracellular NADH concentration has been reported to be about 0.2 mM; with low flow ischemia, levels rise to above 1 mM (42). Compared with xanthine oxidase, AO has a much higher affinity of NADH for nitrite reduction. The K m value of NADH for xanthine oxidase is ϳ0.90 mM, whereas that for AO is almost 40-fold lower with a value of 24 M. Thus, only under ischemic conditions when the levels of NADH rise to Ͼ0.5 mM will NADH serve as an effective electron donor for XOR-mediated nitrite reduction. On the other hand, because NADH has a much higher affinity for AO, it could serve as an efficient electron donor for AO-mediated nitrite reduction even under normoxic conditions (Fig. 6B).
Major sources of aldehydes in tissues include lipid peroxidation, ␣-amino acid oxidation, glycation, biogenic amine metabolism, and ethanol metabolism. Levels of typical aldehydes are in the range of 10 -100 M (43-46). However, under disease conditions or with alcohol ingestion, these levels are further elevated. We have previously measured that the tissue levels of AO are ϳ60 g/g tissue in liver, 35 g/g in lung, and 5.1 g/g in heart with the activity corresponding to 1.8 units/mg enzyme (21,35). In the presence of physiological levels of NADH (100 M) or aldehyde (50 M), the rate of NO generation followed typical Michaelis-Menten kinetics (Fig. 4).
From the reported AO concentrations and kinetic data, AOmediated NO generation in these tissues can be estimated. According to the enzymatic studies in Fig. 4, AO-mediated NO production from 10 M nitrite would be about 0.3 nM/s in the heart, 2.1 nM/s in the lung, and 3.5 nM/s in the liver with 100 M NADH as electron donor. Under ischemic conditions, marked hypoxia and intracellular acidosis occurs, and pH values in tissues, such as the heart, can fall to levels of 6.0 or below (47). With aldehydes, similar to DMAC (50 -100 M), as reducing substrates, this AO-dependent NO generation rate would be ϳ10% higher. Our experiments measuring the effect of pH on AO-mediated nitrite reduction showed that at pH 6.0 an ϳ35% increase occurred compared with pH 7.4 (Table 1). Thus, in the heart, AO-mediated NO generation could approach that of the maximal NO generation from constitutive NO synthase, estimated to be 1.5 nM/s (12,48). Of note, NO synthase requires molecular oxygen as a substrate so that its production of NO would be impaired under the hypoxic or anoxic conditions observed to trigger AO-mediated nitrite reduction. In the liver and lung that express higher AO levels, nitrite-mediated NO generation would be predicted to exceed this level. Our results showed that nitrite reduction in liver was greatly inhibited by the AO inhibitor raloxifene (Fig. 8A). In the presence of physiological tissue levels of nitrite (5 M), liver AO-mediated NO generation activated sGC with a marked increase in cGMP production (Fig. 8B). Under conditions with increased nitrite concentrations, as can occur with pharmacological nitrite supplementation, the magnitude of NO production from this pathway would be further increased, and this increase would be approximately linear with an increase in tissue nitrite concentration.
Several alternative pathways of nitrite-dependent NO generation have been observed occurring in biological systems. NO formation can occur by the simple process of nitrite disproportionation or by a variety of enzyme-catalyzed nitrite reduction pathways (49). XOR-mediated nitrite reduction is among the most potent of these NO generation pathways. According to the kinetic data in this work as well as the known reducing substrate and enzyme levels, it would be predicted that AOmediated NO generation could exceed the NO generation from XOR in the lung and approach that from XOR in the heart and liver under anaerobic conditions. Compared with XOR, AO has a much higher affinity for NADH. Thus, NADH serves as a better electron donor to AO at the flavin site of the enzyme. The binding of NADH to AO could prevent the further binding of oxygen. Therefore, this pathway would also be predicted to better retain its nitrite reduction in the presence of oxygen.
Overall, our studies demonstrate that AO can be an important source of NO generation. Under anaerobic conditions, AO reduces nitrite to NO at the molybdenum site of the enzyme with NADH or aldehyde as electron donor. Under aerobic conditions, AO-mediated nitrite reduction still occurs, but NADH is the preferred substrate. Furthermore, the process of NO generation from nitrite reduction in tissues is regulated by pH, nitrite, reducing substrate concentrations, and tissue oxygenation. This nitrite-derived NO production from AO in tissues could serve as an important alternative source of NO under ischemia, inflammation, or other conditions in which NO production from NO synthase is impaired.