Xanthine Oxidasecatalyzed Nitrite Reduction

Xanthine oxidase (XO) is a ubiquitous enzyme in mammalian cells that plays important roles in both physiological and pathological conditions. It is involved in the catabolism of purine and pyrimidines, oxidizing hypoxanthine to xanthine and xanthine to uric acid. XO also reduces oxygen, to superoxide and hydrogen peroxide production, and is one of the key enzymes responsible for superoxide-mediated cellular injury.

Although it has been established that XO can reduce nitrite to NO [4,5,37], questions remained regarding the biological importance of this pathway of NO production, as well as the mechanism, magnitude, and substrate specificity of this process. It was first reported that NADH, but not xanthine, can act as an electron donor to XO and catalyze nitrite reduction [4,37]. Xanthine or hypoxanthine was found to inhibit this NO formation from XO [37]. However, later it was reported that xanthine can serve as a reducing substrate to stimulate nitrite reduction [38]. In contrast to this, other investigators reported that XO in the presence of xanthine does not reduce nitrite to NO [39]. In addition, in these studies, there were large differences regarding the rates of NO formation and Km values of XO for nitrite or the requisite reducing substrate. In view of these uncertainties, it had not been possible to ascertain the biological relevance and importance of this pathway of NO generation. Therefore, studies using EPR, chemiluminescence NO analyzer, and NO electrode techniques were performed to measure the magnitude and kinetics of NO formation that arise due to XO-mediated nitrite reduction [40]. Data obtained using each of these three methods confirmed that XO does reduce nitrite to NO under anaerobic conditions. Each of the typical reducing substrates xanthine, DBA, and NADH can act as electron donors to support this XO-mediated nitrite reduction (Fig. 2). The results of these studies, along with the inhibition seen with oxypurinol, suggested that reduced XO was the direct electron donor to nitrite, with nitrite binding and reduction occurring at the molybdenum site. Whereas NADH-stimulated NO generation was inhibited by the flavin modifier DPI, NO generation stimulated by xanthine

Fig. 1. Effect of ischemic heart tissue homogenate on the magnitude of NO production from nitrite. (A) Chemi-luminescence measurements were performed with addition of nitrite 1 mM (first arrow) and then 0.1 ml ischemic heart tissue homogenate (second arrow) to 2 ml of phosphate buffer at pH 5.5. Addition of heart homogenate stimulated a marked increase in the rate of NO formation. (B) With addition of heart tissue homogenate prior to nitrite (first arrow), NO generation was not observed; however, after addition of nitrite (second arrow), NO formation occurred identical to that in (A).

Fig. 1. Effect of ischemic heart tissue homogenate on the magnitude of NO production from nitrite. (A) Chemi-luminescence measurements were performed with addition of nitrite 1 mM (first arrow) and then 0.1 ml ischemic heart tissue homogenate (second arrow) to 2 ml of phosphate buffer at pH 5.5. Addition of heart homogenate stimulated a marked increase in the rate of NO formation. (B) With addition of heart tissue homogenate prior to nitrite (first arrow), NO generation was not observed; however, after addition of nitrite (second arrow), NO formation occurred identical to that in (A).

Fig. 2. Measurement of the rate of NO generation from XO-catalyzed nitrite reduction. Measurements were performed using a chemiluminescence NO analyzer under anaerobic conditions at 37°C in PBS, pH 7.4. The arrows show the time at which XO (0.02 mg/ml) was added to A and B. Tracing A shows the data for 1.0 mM nitrite and 5 ^M xanthine, B shows 1.0 mM nitrite in the presence of 1.0 mM NADH, and C shows 1.0 mM nitrite without XO.

Time (seconds)

Fig. 2. Measurement of the rate of NO generation from XO-catalyzed nitrite reduction. Measurements were performed using a chemiluminescence NO analyzer under anaerobic conditions at 37°C in PBS, pH 7.4. The arrows show the time at which XO (0.02 mg/ml) was added to A and B. Tracing A shows the data for 1.0 mM nitrite and 5 ^M xanthine, B shows 1.0 mM nitrite in the presence of 1.0 mM NADH, and C shows 1.0 mM nitrite without XO.

or DBA was unaffected. Thus, whereas xanthine or DBA directly reduces the molybdenum center, NADH initially reduces the flavin, which subsequently transfers electrons to the molybdenum.

Initial studies reported a Km of 22.9 mM for nitrite reduction in the presence of NADH [37]. A subsequent study reported a Km value of 2.4 mM [4]. However, the assessment of the Km by measurement of NADH depletion rather than NO generation reported a Km of 16 mM [4]. We observed that the Km for nitrite was consistently about 2.3 mM in the presence of 1.0 mM NADH. This observation agrees closely with the report of Zhang et al. [4]. In contrast with the studies that report only enzyme reduction by NADH and the study reporting a Km of 36 mM for nitrite in the presence of xanthine, we measured the Km for nitrite is 2.4 ± 0.2 mM for each of the three types of substrates studied, NADH (1 mM), xanthine (5 mM), and 2,3-dihydroxybenz-aldehyde (DBA 40 mM). Possible reasons for variable results could relate to the conditions used for NO purging from the solutions, leak of oxygen into the measurement system, or partial enzyme inactivation or denaturation. Indeed, in the report, Godber et al. [41] acknowledge that phase equilibration and gas flow factors led them to delay their measurements for 2 min after initiation of the reaction. In their system, they measure the spontaneous liberation of NO into the gas headspace and follow steady state conditions with equilibration of the XO in the presence of NO that is generated. Because only a small fraction of the NO generated is used for detection, this approach limits the sensitivity that can be obtained, and therefore, these studies were performed with high nitrite concentrations in the range of 5-120 mM, about 1000 times above typical tissue levels. In our study, the entire

NO generated were rapidly purged into the gas phase and this enabled efficient NO detection and limited NO-mediated effects on the enzyme, enabling detection of the initial rate of the enzyme with physiological/pharmacological nitrite concentrations of 5 ^M to 1 mM. Of note, our parallel measurements of the rates of NO generation performed by NO electrode yielded similar values.

Although xanthine was the highest efficiency reducing substrate of XO-catalyzed nitrite reduction, excessive xanthine exhibited inhibition of NO production. Previous studies reported that enzyme inactivation resulted from NO-induced conversion of XO to its relatively inactive desulfo-form [4]. However, this could not explain why XO kept its activity (95%) when NADH acted as reducing substrate [41]. Also, presence of excess NADH (0.10 mM) or DBA (0.2 mM) had no inhibitive effect on XO-catalyzed NO generation. ONOO-markedly inhibits XO activity in dose-dependent manner, whereas NO from NO gas in concentrations up to 200 mM had no effect [42]. So inactivation of XO [41] could be caused by ONOO- formation triggered upon exposure to oxygen at the time of spectrophotometric activity assay.

The mechanism of NO formation occurs due to nitrite reduction at the molybdenum site, with either NADH or xanthine serving as reducing substrates. Since oxypurinol inhibits substrate binding at the molybdenum site of the enzyme, this suggests that nitrite binds to the reduced Mo site. Diphenyleneiodonium (DPI), which acts at the flavin adenine dinucleotide (FAD) reaction site, inhibited XO-dependent nitrite reduction only when NADH was used as the reducing substrate and it did not inhibit NO generation when xanthine was used (Fig. 3). This suggests that NADH donates electrons to FAD, and then electrons are transported back to

Fig. 3. Effect of site-specific inhibitors on XO-mediated NO formation. The inhibitive effect of oxypurinol, which binds to the molybdenum site, and DPI, which modifies the flavin, were determined for xanthine (X) or NADH-mediated NO generation. For the left set of bars, experiments were performed with 0.5 mM nitrite, 5 m-M xanthine, and 0.02 mg/ml XO, and for the right set of bars, experiments were performed with 1.0 mM nitrite, 1.0 mM NADH, and 0.04 mg/ml XO. Control, without inhibitor; DPI, 20 ^M; oxypurinol, 20 mM.

Fig. 3. Effect of site-specific inhibitors on XO-mediated NO formation. The inhibitive effect of oxypurinol, which binds to the molybdenum site, and DPI, which modifies the flavin, were determined for xanthine (X) or NADH-mediated NO generation. For the left set of bars, experiments were performed with 0.5 mM nitrite, 5 m-M xanthine, and 0.02 mg/ml XO, and for the right set of bars, experiments were performed with 1.0 mM nitrite, 1.0 mM NADH, and 0.04 mg/ml XO. Control, without inhibitor; DPI, 20 ^M; oxypurinol, 20 mM.

reduce the Mo that in turn reduces nitrite to NO. When xanthine or aldehydes are the electron donors, both XO reduction (by xanthine or aldehydes) and oxidation (by nitrite) takes place at the Mo site so that only oxypurinol could inhibit XO-dependent NO formation. Although xanthine is the highest efficiency reducing substrate of XO-catalyzed nitrite reduction, excessive xanthine exhibits inhibition of NO production by binding to the molybdenum site of the reduced enzyme [43,44], thus blocking the binding of nitrite at this enzyme site. This xanthine-mediated inhibition, which has also been demonstrated by Godber et al. [41], may explain the prior failure to detect XO-mediated NO generation from nitrite in studies in which 150 mM xanthine were used [37].

It has been reported that purine and aldehyde substrate hydroxylation takes place via a base-catalyzed mechanism and that substrate must be protonated for hydroxylation [41]. The rate of XO reduction by purine and aldehydes greatly increases when the pH value is increased from 6.0 to 8.0, and this increased rate of XO reduction will lead to an increased rate of nitrite reduction. However, experiments showed that acidic conditions promote XO-catalyzed nitrite reduction. NO generation increased as the pH was decreased from 8.0 down to 7.4 or from 7.4 down to 6.0 (see Table 1), suggesting that nitrite reduction takes place via an acid-catalyzed mechanism, presumably due to nitrite protonation. HNO2 concentration increases when the pH decreases, and it could be the direct binding substrate of XO. Although the decrease of pH would decrease the rate of XO reduction by reducing substrates, it would greatly increase the speed of XO oxidation by nitrite/HNO2.

From the studies performed, it is clear that XO can catalyze the process of NO generation from nitrite under anaerobic or markedly hypoxic conditions similar to those occurring in ischemic tissues. The key questions are, what is the magnitude of this process, and whether the levels of NO produced are likely to have functional significance.

The activity of XO in the postischemic rat heart is 16.8 milliunits/g of protein [17], which corresponds to 0.013 mg of XO/g of protein or ~ 3.4 mg/g of cell water. The total XO and xanthine dehydrogenase (XDH) activity, however, is 10-fold above this value. In the ischemic heart, xanthine levels rise from near zero to values in the order of 10-100 mM, and nitrite levels are ~ 10 mM [1,2,23]. At normal pH values of 7.4, the rate of nitrite degradation due to simple chemical disproportionation is ~0.05 pM/s, as previously reported [35], whereas the rate of XO-catalyzed nitrite reduction would be ~100 pM/s. It has been previously reported in studies from rat heart homogenates that maximally activated nitric oxide synthase produces 1.5 nM/s of NO. Thus xanthine oxidoreductase-mediated NO generation could approach that of the maximal NO production from NOS. Under conditions with increased tissue nitrite concentrations, the magnitude of NO production from this pathway would be further increased; however, it is also clear that with marked elevations in xanthine, it would be inhibited. Because under the acidic and markedly hypoxic conditions

Table 1 Effect of pH on NO generation rate (nmol • mg-1 • s-1) from 1 mM nitrite in the presence of 0.02 mg/ml XO

Table 1 Effect of pH on NO generation rate (nmol • mg-1 • s-1) from 1 mM nitrite in the presence of 0.02 mg/ml XO

pH

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