title: Nitrite reductase activity of rat and human xanthine oxidase, xanthine dehydrogenase, and aldehyde oxidase: evaluation of their contribution to NO formation in vivo.
abstract: Nitrite is presently considered a NO "storage form" that can be made available, through its one-electron reduction, to maintain NO formation under hypoxia/anoxia. The molybdoenzymes xanthine oxidase/dehydrogenase (XO/XD) and aldehyde oxidase (AO) are two of the most promising mammalian nitrite reductases, and in this work, we characterized NO formation by rat and human XO/XD and AO. This is the first characterization of human enzymes, and our results support the employment of rat liver enzymes as suitable models of the human counterparts. A comprehensive kinetic characterization of the effect of pH on XO and AO-catalyzed nitrite reduction showed that the enzyme's specificity constant for nitrite increase 8-fold, while the Km(NO2(-)) decrease 6-fold, when the pH decreases from 7.4 to 6.3. These results demonstrate that the ability of XO/AO to trigger NO formation would be greatly enhanced under the acidic conditions characteristic of ischemia. The dioxygen inhibition was quantified, and the Ki(O2) values found (24.3-48.8 μM) suggest that in vivo NO formation would be fine-tuned by dioxygen availability. The potential in vivo relative physiological relevance of XO/XD/AO-dependent pathways of NO formation was evaluated using HepG2 and HMEC cell lines subjected to hypoxia. NO formation by the cells was found to be pH-, nitrite-, and dioxygen-dependent, and the relative contribution of XO/XD plus AO was found to be as high as 50%. Collectively, our results supported the possibility that XO/XD and AO can contribute to NO generation under hypoxia inside a living human cell. Furthermore, the molecular mechanism of XO/AO-catalyzed nitrite reduction was revised.
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa , 2829-516 Caparica, Portugal.
authors.forename: Luisa B
authors.initials: LB
authors.lastname: Maia
authors.forename: Vânia
authors.initials: V
authors.lastname: Pereira
authors.forename: Lurdes
authors.initials: L
authors.lastname: Mira
authors.forename: José J G
authors.initials: JJ
authors.lastname: Moura
mesh_terms: D042931:Aldehyde Oxidase / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism; D000818:Animals; D055162:Biocatalysis; D042783:Endothelial Cells; D056945:Hep G2 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D055806:Microvessels / Q000166:cytology; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D009572:Nitrite Reductases / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D013997:Time Factors; D019820:Xanthine / Q000378:metabolism; D014968:Xanthine Dehydrogenase / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000378:metabolism*
chemical_list: D000447:Aldehydes; D009573:Nitrites; D009243:NAD; D019820:Xanthine; D009569:Nitric Oxide; D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase; D042931:Aldehyde Oxidase; D009572:Nitrite Reductases; D010100:Oxygen