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NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

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Abstract

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O2 •− source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O2 •− molecule and half a H2O2 molecule per NADH molecule, at rates 3 times those observed for XO (29.2 ± 1.6 and 9.38 ± 0.31 min−1, respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 ± 1.36 μM−1 min−1) was found to be higher than that of the XO specificity constant (1.07 ± 0.09 μM−1 min−1). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O2 •− source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O2 •− than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.

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Abbreviations

AFR:

Activity-to-flavin ratio

AO:

Aldehyde oxidase

EPR:

Electron paramagnetic resonance

FAD:

Flavin adenine dinucleotide

ROS:

Reactive oxygen species

Sim:

Simulated

XD:

Xanthine dehydrogenase

XO:

Xanthine oxidase

XOR:

Xanthine oxidoreductase

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Authors and Affiliations

  1. Centro de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, 1749-016, Lisbon, Portugal

    Luisa Maia, Ana Ponces-Freire & Lurdes Mira

  2. Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, 1749-016, Lisbon, Portugal

    Luisa Maia, Ana Ponces-Freire & Lurdes Mira

  3. REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, 2829-516, Caparica, Portugal

    Rui O. Duarte & José J. G. Moura

  4. Centro de Metabolismo e Endocrinologia da Faculdade de Medicina de Lisboa, Faculdade de Medicina de Lisboa, 1649-016, Lisbon, Portugal

    Lurdes Mira

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  1. Luisa Maia
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  2. Rui O. Duarte
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  5. Lurdes Mira
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Correspondence to Lurdes Mira.

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Maia, L., Duarte, R.O., Ponces-Freire, A. et al. NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production. J Biol Inorg Chem 12, 777–787 (2007). https://doi.org/10.1007/s00775-007-0229-7

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  • DOI: https://doi.org/10.1007/s00775-007-0229-7

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