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. 2009 Sep 28;284(49):33850–33858. doi: 10.1074/jbc.M109.019125

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

Haitao Li 1,1, Tapan Kumar Kundu 1, Jay L Zweier 1,2
PMCID: PMC2797155  PMID: 19801639

Abstract

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.

Introduction

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 (1–5). 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 (11–16). 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 (27–31). The amino acid sequence of AO and XOR are remarkably similar, with ∼86% 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 Km, 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 characterization 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.

EXPERIMENTAL PROCEDURES

Materials

4-(Dimethylamino)cinnamaldehyde (DMAC), NADH, sodium nitrite, diphenyleneiodonium chloride (DPI), and raloxifene hydrochloride were obtained from Sigma. Soluble guanylyl cyclase (sGC) was obtained from Alexis Biochemical Corp. (San Diego). Direct cGMP assay kit was obtained from Assay Designs, Inc. (Ann Arbor, MI), and cGMP production was quantified by immunoassay according to the protocol provided by the company. N-Methyl-d-glucamine dithiocarbamate (MGD) was synthesized using carbon disulfide and N-methyl-d-glucamine (11, 34). Ferrous ammonium sulfate was purchased from Aldrich (99.997%). [15N]Nitrite was obtained from Cambridge Isotope Laboratories, Inc. Dulbecco's phosphate-buffered saline (PBS) and Hanks' buffered salt solution (HBSS) were obtained from Invitrogen. Millipore ultra-free centrifugal filter (nominal molecular weight limit 10,000) was obtained from Fisher.

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 μm 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 ϵ = 30.5 mm−1 cm−1. One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of DMAC/min at 30 °C (35). The activity of freshly isolated AO was ∼1.8 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 (∼0.15 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 (argon-purged) 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 NO2, 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).

Compared with measurements performed with argon (100%), the efficiency of NO measurement with air or 10, 5, 2, and 1% oxygen/nitrogen was 92.0, 92.7, 96.6, 98.8, and 99.2% respectively. Results showed that the chemiluminescence linearly increased with NO generation when purging with any of these gas mixtures (γ2 >99%) (24).

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 μm nitrite, and with 1 ml of reaction buffer (10 ng sGC, 5 mm EDTA, 2 mm MgCl2, 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 × 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 ± 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.

RESULTS

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 NO2, 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 μm) or NADH (100 μm) (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).

FIGURE 1.

FIGURE 1.

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Measurement of the rate of NO generation from AO-catalyzed nitrite reduction. Measurements were performed using a chemiluminescence NO analyzer under anaerobic conditions at 37 °C in 5 ml of PBS, pH 7.4. The arrows show the time at which AO (0.01 mg/ml) was added. A shows the data for 1.0 mm nitrite, 50 μm DMAC with (trace a) or without AO (trace b). B shows the data for 1.0 mm nitrite, 100 μm NADH, with (trace a) or without AO (trace b).

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 Km) 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. 15NO generated from [15N]nitrite is paramagnetic and binds with high affinity to the water-soluble spin trap Fe2+-MGD forming the mononitrosyl iron complex that exhibits a characteristic doublet 15NO-Fe2+-MGD spectrum, rather than the triplet observed with natural abundance 14NO, 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 μm) and DMAC or NADH also no signal was seen (Fig. 2B). However, upon addition of AO, a marked 15NO-Fe2+-MGD signal appeared (Fig. 2C). Over 10 min, 4.8 ± 0.6 μm NO generation was detected with DMAC (Fig. 2C) or 3.2 ± 0.4 μm with NADH (Fig. 2D) as electron donor. Thus, typical AO-reducing substrates were able to act as electron donors for AO-catalyzed nitrite reduction and triggered large levels of NO generation under anaerobic conditions.

FIGURE 2.

FIGURE 2.

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EPR measurement of NO generated from nitrite under anaerobic conditions. Spectra are shown of the (MGD)2-Fe2+-15NO adducts formed in solutions of 100 μm (MGD)2-Fe2+ complex in PBS, pH 7.4, after 10 min with the following: A, 0.04 mg/ml AO, 50 μm DMAC; B, 50 μm DMAC, 100 μm [15N]nitrite; C, 0.04 mg/ml AO, 50 μm DMAC, and [15N]nitrite (100 μm); and D, 0.04 mg/ml AO, 100 μm NADH, and [15N]nitrite (100 μm) in 5 ml of PBS, pH 7.4, at 37 °C under anaerobic conditions.

Further measurements of NO generation from pathophysiological levels of nitrite with DMAC and NADH as reducing substrates were performed using a chemiluminescence NO analyzer. With nitrite alone (100 μm) or nitrite in the presence of DMAC (50 μm) or NADH (100 μm), no measurable rate of NO generation was observed (Fig. 3, trace d). However, with the addition of AO in the presence of molybdenum site-binding reducing substrate DMAC (50 μm) (Fig. 3A) or flavin site electron donor NADH (100 μm) (Fig. 3B), prominent NO formation was triggered from 10, 50, or 100 μm nitrite under anaerobic conditions (Fig. 3).

FIGURE 3.

FIGURE 3.

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NO generation from pathophysiological levels of nitrite. Measurements were performed using a chemiluminescence NO analyzer under anaerobic conditions at 37 °C in 5 ml of PBS, pH 7.4. The arrows show the time at which AO (0.01 mg/ml) was added. With 50 μm DMAC (A) or 100 μm NADH (B) as reducing substrate, NO formation rates are shown from 100 μm nitrite (trace a), 50 μm nitrite (trace b), 10 μm nitrite (trace c), and without nitrite (trace d).

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 μm NADH (Fig. 4A) or 50 μm DMAC (Fig. 4B) as electron donor. Typical Michaelis-Menten kinetics were observed as a function of nitrite concentration, and the apparent Km and Vmax 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 AO-catalyzed nitrite reduction. Again, typical Michaelis-Menten kinetics were observed (Fig. 4, C and D), and the apparent values of Km and Vmax 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.

FIGURE 4.

FIGURE 4.

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Kinetics of NO generation from AO as a function of nitrite or reducing substrate concentration. Initial rates of NO generation were measured by chemiluminescence NO analyzer as described in Fig. 1. A shows the rate of NO generation by 0.01 mg/ml AO and 100 μm NADH in the presence of 0.01–10 mm nitrite. B shows the rates of NO generation by 0.01 mg/ml AO and 50 μm DMAC in the presence of 0.01–10 mm nitrite. C shows the rates of NO generation by 0.01 mg/ml AO and 1 mm nitrite in the presence of 10–200 μm NADH. D shows the rates of NO generation by 0.01 mg/ml AO and 1 mm nitrite in the presence of 2–60 μm DMAC. For each of these graphs, the Km and Vmax, values were obtained from corresponding fits (solid line) using the Michaelis-Menten equation with a correlation coefficient of γ2 > 0.97.

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.

FIGURE 5.

FIGURE 5.

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Effect of site-specific inhibitors on AO-mediated NO formation. Rates of NO generation were measured by chemiluminescence NO analyzer as described in Fig. 2. For the left set of bars, experiments were performed with 100 μm nitrite, 50 μm DMAC, and 0.01 mg/ml AO, and for the right set of bars, experiments were performed with 100 μm nitrite, 100 μm NADH, and 0.01 mg/ml AO. The inhibitive effects of raloxifene, which binds to the molybdenum site, and DPI, which modifies the flavin, were determined for DMAC or NADH-mediated NO generation. NO generation rates are shown as follows: A, control (without inhibitor); B, with 100 μm DPI; and C, 10 μm raloxifene.

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 μm 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.

TABLE 1.

Effect of pH on NO generation from nitrite reduction

pH 5.0 pH 6.0 pH 7.4 pH 8.0
NO generation rate (nmol/s·unit)a
DMAC (50 μm) 0.17 ± 0.01 0.56 ± 0.09 0.41 ± 0.06 0.35 ± 0.04
NADH (100 μm) 0.15 ± 0.01 0.46 ± 0.11 0.34 ± 0.05 0.28 ± 0.03
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a The maximal NO generation rates were measured by chemiluminescence NO analyzer as described in Fig. 2. Measurements were performed with 0.01 mg/ml AO in the presence of 100 μm nitrite and reducing substrate under anaerobic conditions at 37 °C. Values are mean ± S.D.

Effect of Oxygen Tension on AO-mediated 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 AO-catalyzed 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 μm, 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 Vmax = 13.5 nmol·s−1·units−1 and Km = 3.3 mm as shown in Fig. 4B, the inhibition constant Ki of oxygen on AO-mediated nitrite reduction was calculated to be 3.8 μm, as obtained from fitting to Equation 1 (γ2 > 0.95),

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FIGURE 6.

FIGURE 6.

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Effect of oxygen on AO-mediated NO generation. A shows the NO formation from AO (0.02 mg/ml) with nitrite (1 mm) and DMAC (50 μm) as reducing substrate. From the fitting (solid line), the Ki value was obtained using the equation (Eq. 1) as noted in the text. B shows the NO formation rates from AO (0.01 mg/ml) with nitrite (1 mm) and NADH (100 μm) as reducing substrate. The oxygen concentrations of 214, 102, 51, 20, 10, and 0 mm, respectively, were achieved with purging of the sample solution at 37 °C with air, 10, 5, 2, or 1% oxygen or argon.

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 ± 0.34, 2.47 ± 0.24, 2.13 ± 0.17, 1.47 ± 0.15, 1.27 ± 0.11, and 1.13 ± 0.09 nmol·s−1·units−1, respectively (Fig. 6B). The rate of NO generation decreased with the increase of pO2 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 μm) with NADH or DMAC in the presence or absence of AO in the reaction buffer (10 ng of sGC, 5 mm EDTA, 2 mm MgCl2, 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 μm nitrite and 100 μm NADH or 50 μm 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 μm) with either NADH (100 μm) or DMAC (50 μm) as electron donor (Fig. 7).

FIGURE 7.

FIGURE 7.

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Soluble guanylyl cyclase activation by AO-mediated nitrite reduction. The reaction buffer is PBS (1 ml, pH 7.4) with EDTA (5 mm), MgCl2 (2 mm), sGC (10 ng), and GTP (1 mm). Bar A, nitrite (10 μm) and DMAC (50 μm); bar B, AO (0.01 mg/ml), nitrite (10 μm), and DMAC (25 μm); bar C, nitrite (10 μm) and NADH (100 μm); bar D, AO (0.01 mg/ml), nitrite (10 μm) and NADH (100 μm). Incubation was carried out for 10 min in the reaction buffer under anaerobic conditions. sGC activation was determined from the measurements of cGMP formation.

NO Generation and sGC Activation from Rat Liver

To further access the importance of AO-mediated 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 ∼53% (Fig. 8A), and cGMP formation was inhibited ∼49% (Fig. 8B).

FIGURE 8.

FIGURE 8.

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NO generation and soluble guanylyl cyclase activation by nitrite in liver. A shows NO generation from 1 mm nitrite in liver tissue homogenate (8 mg/ml protein) in 5 ml of HBSS. Bar a is without and bar b is with the AO inhibitor raloxifene (1 μm). B shows cGMP formation triggered by 5 μm nitrite in liver tissue homogenate. The reactions were performed with liver homogenate (10 mg/ml protein) in HBSS (1 ml, pH 7.4) with EDTA (5 mm), MgCl2 (2 mm), sGC (10 ng), and GTP (1 mm). Bar a is without and bar b is with the AO inhibitor raloxifene (1 μm). After 5 min of incubation under anaerobic conditions, cGMP formation was detected as described under “Experimental Procedures.”

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 site-reducing substrate NADH and molybdenum site-reducing substrate DMAC could act as electron donors to support this AO-mediated 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.

SCHEME 1.

SCHEME 1.

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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 AO-catalyzed 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,

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where Eox is the fully oxidized enzyme; Ered is the two-electron reduced enzyme, and E′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 that for each aldehyde or NADH oxidized, two molecules of nitrite can be reduced to NO. The total enzyme concentration, [Et], can be defined as shown in Equation 2,

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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,

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where terms are defined as shown in Equations 4 and 5,

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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 Km of nitrite for AO is ∼3 mm, which far exceeds normal cellular levels, which are ∼10 μm (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,

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With molybdenum site electron donor DMAC as reducing substrate, AO-mediated NO generation is greatly inhibited by oxygen. Oxygen acts as a competitive inhibitor (Ki ∼ 3.8 μm) 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 Km value of ∼24 μm 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+ is reduced, and NADH concentrations will be increased. In rat liver cytosol, the total amount of NADH was estimated to be ∼270 μm, and the total amount of NAD+ and NADH is ∼1 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 Km value of NADH for xanthine oxidase is ∼0.90 mm, whereas that for AO is almost 40-fold lower with a value of 24 μm. Thus, only under ischemic conditions when the levels of NADH rise to >0.5 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, α-amino acid oxidation, glycation, biogenic amine metabolism, and ethanol metabolism. Levels of typical aldehydes are in the range of 10–100 μm (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 ∼60 μg/g tissue in liver, 35 μg/g in lung, and 5.1 μg/g in heart with the activity corresponding to 1.8 units/mg enzyme (21, 35). In the presence of physiological levels of NADH (100 μm) or aldehyde (50 μm), the rate of NO generation followed typical Michaelis-Menten kinetics (Fig. 4).

From the reported AO concentrations and kinetic data, AO-mediated NO generation in these tissues can be estimated. According to the enzymatic studies in Fig. 4, AO-mediated NO production from 10 μm 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 μm 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 μm), as reducing substrates, this AO-dependent NO generation rate would be ∼10% higher. Our experiments measuring the effect of pH on AO-mediated nitrite reduction showed that at pH 6.0 an ∼35% 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 μm), 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 AO-mediated 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.

*

This work was supported, in whole or in part, by National Institutes of Health Grants HL63744, HL65608, and HL38324 (to J. L. Z.). This work was also supported by the Ohio Valley Affiliate of the American Heart Association Grant BGIA-0565249B (to H. L.).

3
The abbreviations used are:
NO
nitric oxide
AO
aldehyde oxidase
XOR
xanthine oxidoreductase
sGC
soluble guanylyl cyclase
DMAC
4-(dimethylamino)cinnamaldehyde
MGD
N-methyl-d-glucamine dithiocarbamate
DPI
diphenyleneiodonium chloride
PBS
phosphate-buffered saline
HBSS
Hanks' buffered salt solution.

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