Nitrite reductase activity of rat and human xanthine oxidase, xanthine dehydrogenase, and aldehyde oxidase: evaluation of their contribution to NO formation in vivo

Biochemistry. 2015 Jan 27;54(3):685-710. doi: 10.1021/bi500987w. Epub 2015 Jan 8.


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.

MeSH terms

  • Aldehyde Oxidase / metabolism*
  • Aldehydes / metabolism
  • Animals
  • Biocatalysis
  • Endothelial Cells
  • Hep G2 Cells
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Male
  • Microvessels / cytology
  • Models, Molecular
  • NAD / metabolism
  • Nitric Oxide / metabolism*
  • Nitrite Reductases / metabolism*
  • Nitrites / metabolism
  • Oxidation-Reduction
  • Oxygen / metabolism
  • Rats, Sprague-Dawley
  • Substrate Specificity
  • Time Factors
  • Xanthine / metabolism
  • Xanthine Dehydrogenase / metabolism*
  • Xanthine Oxidase / metabolism*


  • Aldehydes
  • Nitrites
  • NAD
  • Xanthine
  • Nitric Oxide
  • Xanthine Dehydrogenase
  • Xanthine Oxidase
  • Aldehyde Oxidase
  • Nitrite Reductases
  • Oxygen