Characterization of the Effects of Oxygen on Xanthine Oxidase-mediated Nitric Oxide Formation

Abstract

Under anaerobic conditions, xanthine oxidase (XO)-catalyzed nitrite reduction can be an important source of nitric oxide (NO). However, questions remain regarding whether significant XO-mediated NO generation also occurs under aerobic conditions. Therefore, electron paramagnetic resonance, chemiluminescence NO-analyzer, and NO-electrode studies were performed to characterize the kinetics and magnitude of XO-mediated nitrite reduction as a function of oxygen tension. With substrates xanthine or 2,3-dihydroxybenz-aldehyde that provide electrons to XO at the molybdenum site, the rate of NO production followed Michaelis-Menten kinetics, and oxygen functioned as a competitive inhibitor of nitrite reduction. However, with flavin-adenine dinucleotide site-binding substrate NADH as electron donor, aerobic NO production was maintained at more than 70% of anaerobic levels, and binding of NADH to the flavin-adenine dinucleotide site seemed to prevent oxygen binding. Therefore, under aerobic conditions, NADH would be the main electron donor for XO-catalyzed NO production in tissues. Studies of the pH dependence of NO formation indicated that lower pH values decrease oxygen reduction but greatly increase nitrite reduction, facilitating NO generation. Isotope tracer studies demonstrated that XO-mediated NO formation occurs in normoxic and hypoxic heart tissue. Thus, XO-mediated NO generation occurs under aerobic conditions and is regulated by oxygen tension, pH, nitrite, and reducing substrate concentrations. Under anaerobic conditions, xanthine oxidase (XO)-catalyzed nitrite reduction can be an important source of nitric oxide (NO). However, questions remain regarding whether significant XO-mediated NO generation also occurs under aerobic conditions. Therefore, electron paramagnetic resonance, chemiluminescence NO-analyzer, and NO-electrode studies were performed to characterize the kinetics and magnitude of XO-mediated nitrite reduction as a function of oxygen tension. With substrates xanthine or 2,3-dihydroxybenz-aldehyde that provide electrons to XO at the molybdenum site, the rate of NO production followed Michaelis-Menten kinetics, and oxygen functioned as a competitive inhibitor of nitrite reduction. However, with flavin-adenine dinucleotide site-binding substrate NADH as electron donor, aerobic NO production was maintained at more than 70% of anaerobic levels, and binding of NADH to the flavin-adenine dinucleotide site seemed to prevent oxygen binding. Therefore, under aerobic conditions, NADH would be the main electron donor for XO-catalyzed NO production in tissues. Studies of the pH dependence of NO formation indicated that lower pH values decrease oxygen reduction but greatly increase nitrite reduction, facilitating NO generation. Isotope tracer studies demonstrated that XO-mediated NO formation occurs in normoxic and hypoxic heart tissue. Thus, XO-mediated NO generation occurs under aerobic conditions and is regulated by oxygen tension, pH, nitrite, and reducing substrate concentrations. Nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; NOS, nitric oxide synthase; XO, xanthine oxidase; FAD, flavin-adenine dinucleotide; DBA, 2,3-dihydroxybenz-aldehyde; DPI, diphenyleneiodonium chloride; MGD, N-methyl-d-glucamine dithiocarbamate; PBS, phosphate-buffered saline; HBSS, Hanks' balanced salt solution; SOD, superoxide dismutase; XOR, xanthine oxidoreductase. is a free radical endogenously produced in biological tissues and is an important regulator of numerous biological functions (1Palmer R.M. Ferrige A.G. Moncada S. Nature. 1987; 327: 524-526Crossref PubMed Scopus (9364) Google Scholar, 2Ignarro L.J. Buga G.M. Wood K.S. Byrns R.E. Chaudhuri G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 9265-9269Crossref PubMed Scopus (4362) Google Scholar, 3Furchgott R.F. Vanhoutte P.M. FASEB J. 1989; 3: 2007-2018Crossref PubMed Scopus (1688) Google Scholar, 4Moncada S. Palmer R.M. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar). NO can also cause cellular injury by means of reaction with superoxide to form the potent oxidant peroxynitrite (5Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6730) Google Scholar, 6Beckman J.S. Nature. 1990; 345: 27-28Crossref PubMed Scopus (136) Google Scholar, 7Wang P. Zweier J.L. J. Biol. Chem. 1996; 271: 29223-29230Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). Specific nitric oxide synthase (NOS) enzymes have been generally considered to be the primary source of NO in biological systems. These enzymes metabolize arginine to citrulline with the formation of NO, and oxygen is required for this process (8Bredt D.S. Snyder S.H. Neuron. 1992; 8: 3-11Abstract Full Text PDF PubMed Scopus (1840) Google Scholar, 9Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2173) Google Scholar). Under severe hypoxic conditions, such as those that occur in ischemic tissues, the NO production from NOS is impaired because of the lack of oxygen. In ischemic tissues, other factors including marked acidosis also impair the function of NOS. More recently, it has been demonstrated that there is another enzymatic pathway of NO generation that does not require oxygen. It has been shown that under anaerobic conditions, XO-mediated nitrite or nitrate reduction can be a prominent source of NO (10Zhang Z. Naughton D. Winyard P.G. Benjamin N. Blake D.R. Symons M.C. Biochem. Biophys. Res. Commun. 1998; 249: 767-772Crossref PubMed Scopus (218) Google Scholar, 11Millar T.M. Stevens C.R. Benjamin N. Eisenthal R. Harrison R. Blake D.R. FEBS Lett. 1998; 427: 225-228Crossref PubMed Scopus (414) Google Scholar, 12Godber B.L. Doel J.J. Sapkota G.P. Blake D.R. Stevens C.R. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 14Li H. Samouilov A. Liu X. Zweier J.L. Biochemistry. 2003; 42: 1150-1159Crossref PubMed Scopus (158) Google Scholar). This pathway is also enhanced under the acidotic conditions that occur during ischemia. Such XO-mediated NO generation can serve as an alternative source of NO under the near anoxic conditions that occur with no-flow ischemia (13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 14Li H. Samouilov A. Liu X. Zweier J.L. Biochemistry. 2003; 42: 1150-1159Crossref PubMed Scopus (158) Google Scholar). Xanthine oxidase (XO) is a flavoprotein enzyme that is widely distributed in various mammalian tissues. In addition to its FAD binding site, it also has molybdenum and iron sulfur centers. XO plays important roles in both physiological and pathological conditions. It is well known as an important source of superoxide and reactive oxygen species generation (15Zweier J.L. Kuppusamy P. Lutty G.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4046-4050Crossref PubMed Scopus (561) Google Scholar). More recently, XO-mediated nitrite reduction to NO has been reported under anaerobic conditions. The magnitude and kinetics of this process have been characterized, and it has been shown that under conditions occurring during noflow ischemia, myocardial XO and nitrite levels are sufficient to generate NO at levels comparable with or exceeding those from maximally activated nitric oxide synthases (13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 14Li H. Samouilov A. Liu X. Zweier J.L. Biochemistry. 2003; 42: 1150-1159Crossref PubMed Scopus (158) Google Scholar). XO has different site-specific electron donors. Xanthine and 2,3-dihydroxybenz-aldehyde (DBA) serve as electron donors to XO by binding to the molybdenum site, the site at which XO-mediated nitrite reduction occurs (13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). However, NADH reduces XO at the FAD site of the enzyme, which is the site that also reduces oxygen. Previous studies on XO-mediated nitrite reduction under anaerobic conditions showed that xanthine and DBA are more effective electron donors than NADH (13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Under aerobic conditions, it is unclear whether significant XO-mediated NO generation would occur because oxygen would be expected to effectively compete with nitrite for reduction by the enzyme. In addition, under aerobic conditions, substrate preference and control could be very different from that in the absence of oxygen. Thus, a number of questions remain regarding the magnitude and mechanism of XO-mediated NO generation in the presence of oxygen and the significance of this process in aerobic biological systems. To investigate the mechanism and effect of oxygen on XO-mediated nitrite reduction, electron paramagnetic resonance (EPR) chemiluminescence NO and NO studies were performed to characterize the kinetics and magnitude of XO-mediated nitrite reduction as a function of oxygen tension. that XO-mediated NO production does occur under aerobic oxygen can of this NO generation and greatly the of It was demonstrated in normoxic heart that XO-mediated NO generation can be oxidase from oxygen superoxide superoxide diphenyleneiodonium nitrite, dinucleotide and (DBA) were from N-methyl-d-glucamine was and was from was from were performed a at were performed a at with a of of and of for of NO were as The reaction for nitrite reduction was in and the NO was by of or oxygen of known to was with the iron of MGD, which the that a at and were by and to of in the with in This was to the reaction in the from the and by the reaction of with nitrite or with the enzyme S. Res. PubMed Scopus Google Scholar, H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). from were at for of NO formation and was performed by of the with to that from a were by of known of NO with to which the and of the as the rate of NO production was a nitric oxide a to to a In the NO was with which that was by a of and of NO from the reaction were at a in a with and which the of the of by levels at the by means of of in and The of NO was by of the from the and in The of NO was from the of the with that from nitrite to A. Kuppusamy P. Zweier J.L. Biochem. Biophys. 1998; PubMed Scopus Google Scholar). The of nitrite in the was by reduction to NO and to values from nitrite A. Kuppusamy P. Zweier J.L. Biochem. Biophys. 1998; PubMed Scopus Google Scholar). of the NO was performed of studies were performed to the of with different with performed with the of NO with or was or showed that the chemiluminescence with NO generation with of of NO generation by XO were at in an a and a NO The the the which was to the NO in the The was and the with known of NO, on of NO of NO from were with of and with at a of The were and and with to and This heart was in with and with and with a The reaction was in a and maintained at of NO was performed as the shown in are as of at significance of was by a was considered to XO-mediated NO from under is paramagnetic and with to the a iron with at with the of the of NO generation can be performed Zweier J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J.L. P. Samouilov A. Kuppusamy P. PubMed Scopus Google Scholar, N. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). Under aerobic conditions, XO can the reduction of to the formation of superoxide which can with nitric oxide To prevent the of superoxide with the of NO was to the in the NO by XO-catalyzed nitrite reduction was from to the of XO and its reducing substrate xanthine to In the absence of SOD, of XO with nitrite and xanthine also to In the presence of of XO with nitrite and its reducing substrates DBA xanthine or NADH a NO was and Thus, substrates including and xanthine as electron donors for XO-catalyzed nitrite reduction and NO generation under aerobic conditions. reported under anaerobic conditions xanthine is the effective reducing NADH is a effective electron donor for XO-mediated nitrite reduction to NO (13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). However, under aerobic conditions, NADH XO-mediated nitrite reduction occurs with a magnitude of NO generation than that by xanthine or DBA NO were performed to the of NO generation from XO-mediated nitrite reduction under aerobic conditions. to the addition of XO, NO generation was from nitrite in the presence of with xanthine or NADH as reducing However, addition of XO NO generation was from xanthine or NADH and in the presence of Under aerobic conditions, the magnitude and rate of NO generation in the presence of NADH was than that with xanthine In under anaerobic conditions, the magnitude of NO generation in the presence of of xanthine was than that with NADH (13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Thus, both and NO studies demonstrated that NADH is the effective electron donor for XO-mediated NO generation under aerobic conditions. To the rate of XO-mediated NO studies were performed with a chemiluminescence NO NO was from the by It was that the rate of NO generation is than the rate of NO generation by xanthine or DBA under aerobic conditions of XO-catalyzed Nitric in the of of the reducing substrates of XO can NO formation from nitrite under aerobic conditions as well as under anaerobic which is the effective reducing substrate under anaerobic conditions, was the potent reducing substrate in the presence of oxygen. To the effect and investigate the mechanism of nitrite reduction, chemiluminescence of the rate of NO production were performed different site-specific reducing substrates under different oxygen NO formation from XO-catalyzed nitrite reduction was with with or to oxygen in at of and xanthine and DBA provide electrons to XO at the molybdenum site, the site of nitrite binding to the enzyme. xanthine or DBA as reducing the rate of XO-mediated NO formation was as a function of nitrite and Michaelis-Menten kinetics were the of by oxygen and demonstrated that oxygen is a competitive inhibitor to nitrite in XO-mediated nitrite reduction and With xanthine as reducing the values of nitrite, from to the Michaelis-Menten from and to with the increase of with oxygen or values at With DBA as reducing the values for nitrite from and to with the increase of values at NADH with XO at the FAD site of the enzyme. With NADH as electron donor and with NO generation from nitrite not Michaelis-Menten kinetics NO generation from nitrite were maintained at more than 70% of anaerobic levels nitrite with XO NADH and for in or oxygen or in the of NO generation were and or The rate of NO generation with the increase of NADH was the reducing in prominent NO production was of on XO-mediated NO under of site-specific of XO were to investigate the reaction in the process of the XO-catalyzed nitrite reduction under aerobic conditions. to the molybdenum site of the of oxygen it was that the NO generation by DBA, or of the FAD site-specific DPI, XO-mediated NO with NADH as reducing with xanthine or DBA as reducing NO generation was more than This that of the by the binding and of electrons to oxygen by XO, greatly XO-mediated oxygen reduction. Thus, oxygen reduction by the enzyme and in greatly its competitive reaction of nitrite reduction. of pH on XO-catalyzed NO the NO formation under different physiological or pathological conditions and to characterize the mechanism of XO-catalyzed nitrite reduction, were performed to the effect of different pH values on the magnitude of XO-mediated NO generation under aerobic conditions. shown in it was for of the reducing and that pH is the of NO generation. Under aerobic conditions, NO generation more than as the pH from to and another as pH from to of pH on NO generation rate under aerobic generation in a XO-mediated NO from in mammalian under normoxic conditions, from to with values of in and in the myocardial levels to or lower S. S. S. D. N. A. Res. 2003; PubMed Scopus Google Scholar, J.L. Samouilov A. Kuppusamy P. Biophys. PubMed Scopus Google Scholar). To whether nitrite can be to form NO in tissues and to the of XO in this process under aerobic conditions, studies were performed in heart in the presence of as under and heart was in and with The was to formation during This also to the concentrations. was to prevent NO generation from NO production from in the heart was by a than the with and of NO In the absence of was from nitrite with of and However, addition of of heart a was With the addition of the XO inhibitor the was more than In the presence of the of NO generation was more than than that at Thus, XO-mediated nitrite reduction can be a source of NO in heart under conditions of and it is with It has been reported that XO reduction of nitrite to NO under hypoxic conditions (10Zhang Z. Naughton D. Winyard P.G. Benjamin N. Blake D.R. Symons M.C. Biochem. Biophys. Res. Commun. 1998; 249: 767-772Crossref PubMed Scopus (218) Google Scholar, 11Millar T.M. Stevens C.R. Benjamin N. Eisenthal R. Harrison R. Blake D.R. FEBS Lett. 1998; 427: 225-228Crossref PubMed Scopus (414) Google Scholar, 12Godber B.L. Doel J.J. Sapkota G.P. Blake D.R. Stevens C.R. Eisenthal R. Harrison R. J. Biol. Chem. 2000; 275: 7757-7763Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 14Li H. Samouilov A. Liu X. Zweier J.L. Biochemistry. 2003; 42: 1150-1159Crossref PubMed Scopus (158) Google and the kinetics of this process have been It is that XO-mediated nitrite and nitrate reduction occurs and can be an important source of NO, under conditions of and acidosis (13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 14Li H. Samouilov A. Liu X. Zweier J.L. Biochemistry. 2003; 42: 1150-1159Crossref PubMed Scopus (158) Google Scholar). However, questions regarding whether XO-mediated NO generation also occurs in the presence of and the and of this process in biological not been Therefore, performed a of studies chemiluminescence NO and NO to the magnitude and kinetics of XO-mediated NO formation under different oxygen of that XO does nitrite to NO under aerobic conditions. reducing substrates of XO NO generation from XO-mediated nitrite reduction the kinetics of XO-mediated NO formation are different in the presence of binding substrates xanthine or DBA, with that in the presence of the binding substrate NADH and With xanthine or DBA as reducing substrates that electrons to XO at the molybdenum site of enzyme, the rate of NO production followed Michaelis-Menten kinetics, and studies that oxygen as a competitive inhibitor of nitrite reduction The can the in the reaction is the enzyme, is the enzyme, and is the enzyme. to the molybdenum reducing substrates of XO as xanthine and and is the It be for xanthine of nitrite can be to The enzyme can be as the rate of NO generation can be and this can be in the form of the Michaelis-Menten are as It was that a of nitrite a to the the rate of NO generation from XO in the presence of reducing substrates xanthine or DBA under different oxygen the of nitrite for XO with of the These the competitive effect of oxygen on XO-mediated nitrite reduction binding reducing xanthine or DBA, are the electron donors. Under aerobic conditions, with xanthine or DBA as reducing XO-mediated NO production is than the of NO production under anaerobic conditions (13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). With the binding reducing as electron donor, XO-mediated NO production is maintained at more than 70% of the anaerobic levels, and the XO-catalyzed NO generation rate from under anaerobic conditions to under aerobic conditions in the presence of the enzyme and substrate of the with NADH under aerobic conditions, it was that this XO-mediated nitrite reduction not Michaelis-Menten NADH as electron donor to XO at the FAD site, the site as that for oxygen nitrite reduction at the molybdenum site of the enzyme (13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). With NADH as reducing the XO-mediated NO generation occur as shown In XO is in the With FAD site both oxygen and nitrite can electrons from Thus, under aerobic conditions, oxygen is a competitive inhibitor to XO-mediated nitrite reduction in NO generation would decrease greatly in the presence of and it would the kinetics as shown in However, in the FAD site is by the binding of oxygen reduction is at the molybdenum site, XO-mediated nitrite reduction is Thus, the rate of XO-mediated nitrite reduction would be in the presence or absence of oxygen as in The kinetics of this process of XO-mediated nitrite reduction, in the absence of have been (13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Under aerobic conditions, than of the nitrite of XO is which that nitrite reduction the FAD site is with NADH is for the and is in of NADH of and heart of NADH of Scholar). With binding electron donors xanthine or DBA, nitrite reduction is greatly by the presence of with XO-mediated NO generation at more than 70% of anaerobic The of NADH in biological and its on the binding of oxygen that NADH would be the electron donor for XO-catalyzed NO production under aerobic conditions. can the rate of XO-mediated NO production that would occur from nitrite reduction in the heart under physiological conditions. In myocardial NADH has been reported to be to an of with ischemia, levels to Scholar). oxygen levels are in the heart S. S. S. D. N. A. Res. 2003; PubMed Scopus Google Scholar). Xanthine is in the heart at levels of Zweier J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). nitrite levels of have been in the heart J.L. P. Samouilov A. Kuppusamy P. PubMed Scopus Google Scholar). The process of XO-mediated nitrite reduction with NADH as substrate has a for nitrite of (13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). this nitrite the NO formation rate would with nitrite Therefore, under aerobic conditions in the the rate of NO generation would be to be of the rate of NO generation from under anaerobic conditions. The of site-specific were to investigate the reaction site in the process of XO-mediated nitrite reduction under aerobic conditions. DPI, the inhibitor of FAD greatly NO generation under aerobic conditions with xanthine or DBA used as reducing substrate It is known that the binding of DBA, and nitrite, the reduction of XO by With xanthine or DBA as reducing the presence of XO-mediated oxygen reduction at the FAD and the of the enzyme for nitrite reduction at the molybdenum site the reduction of nitrite and the of xanthine and DBA on the molybdenum site of Thus, the or of the FAD site of XO the binding of oxygen and this enzyme from oxygen radical generation to nitric oxide generation in the presence of nitrite and the substrates xanthine or The of in NO generation from XO be is used in biological of nitrite are Previous studies demonstrated that XO-mediated nitrite reduction by means of an pH from to NO generation rate under anaerobic conditions (13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). 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Full Text Full Text PDF PubMed Scopus Google Scholar). substrate and nitrite as well as pH XO-mediated NO production under aerobic conditions, and the biological of this production are by and These that under aerobic conditions, NADH would be the main electron donor for XO-catalyzed NO production in mammalian and tissues. ischemia, the myocardial can increase more than G.M. PubMed Scopus Google xanthine levels can to the of with nitrite levels of Zweier J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J.L. P. Samouilov A. Kuppusamy P. PubMed Scopus Google the oxygen and acidosis greatly XO-mediated NO generation and superoxide The magnitude of XO-mediated NO generation can that of the NO production from NOS (13Li H. Samouilov A. Liu X. Zweier J.L. J. Biol. Chem. 2001; 276: 24482-24489Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). with to levels of as can occur with or ischemia in the presence of this process would be This could NO to and a during ischemia. the NO would with a of peroxynitrite production that can and cellular injury P. Zweier J.L. J. Biol. Chem. 1996; 271: 29223-29230Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). Thus, XO-mediated NO generation occurs under aerobic conditions as well as under anaerobic conditions. With substrates such as xanthine or DBA that at the molybdenum site of the enzyme, oxygen as a competitive inhibitor of nitrite reduction, with which at the FAD site, oxygen a of nitrite reduction. This process of aerobic XO-mediated NO generation is by oxygen tension, pH, nitrite levels, and reducing substrate concentrations. It would be expected to be important under conditions, levels of nitrite in tissues, and under conditions such as or ischemia, cellular acidosis and occur with levels of NADH and other XO