Abstract
Mammalian xanthine oxidase (XO) and Desulfovibrio gigas aldehyde oxidoreductase (AOR) are members of the XO family of mononuclear molybdoenzymes that catalyse the oxidative hydroxylation of a wide range of aldehydes and heterocyclic compounds. Much less known is the XO ability to catalyse the nitrite reduction to nitric oxide radical (NO). To assess the competence of other XO family enzymes to catalyse the nitrite reduction and to shed some light onto the molecular mechanism of this reaction, we characterised the anaerobic XO- and AOR-catalysed nitrite reduction. The identification of NO as the reaction product was done with a NO-selective electrode and by electron paramagnetic resonance (EPR) spectroscopy. The steady-state kinetic characterisation corroborated the XO-catalysed nitrite reduction and demonstrated, for the first time, that the prokaryotic AOR does catalyse the nitrite reduction to NO, in the presence of any electron donor to the enzyme, substrate (aldehyde) or not (dithionite). Nitrite binding and reduction was shown by EPR spectroscopy to occur on a reduced molybdenum centre. A molecular mechanism of AOR- and XO-catalysed nitrite reduction is discussed, in which the higher oxidation states of molybdenum seem to be involved in oxygen-atom insertion, whereas the lower oxidation states would favour oxygen-atom abstraction. Our results define a new catalytic performance for AOR—the nitrite reduction—and propose a new class of molybdenum-containing nitrite reductases.
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Notes
The pterin–molybdenum cofactor is still commonly referred to as “molybdopterin”, because, historically, the cofactor was first identified in molybdenum-containing enzymes. However, the same cofactor molecule is used to coordinate tungsten in some organisms that are able to utilise that element. To avoid confusion between molybdenum and tungsten-containing enzymes, the denomination “pterin–molybdenum cofactor”, proposed by the Nomenclature Committee of the International Union of Biochemistry (http://www.chem.qmul.ac.uk/iubmb/etp/) was chosen.
Xanthine and uric acid represented in neutral form.
For simplicity, xanthine (pK a of 0.8 and 7.4) is represented in the neutral form. Urate (pK a of 5.2 and 11.3) or carboxylate (pK a of 4.2 for benzoic acid) is depicted in the physiological monoanionic form.
Abbreviations
- AOR:
-
Aldehyde oxidoreductase
- DMSOR:
-
Dimethylsulfoxide reductase
- EPR:
-
Electron paramagnetic resonance
- Fe/S:
-
Iron–sulfur centre
- Fe/S–NO:
-
Dinitrosyl–iron–sulfur complex
- (MGD)2–Fe:
-
Ferrous complex of di(N-methyl-d-glucamine dithiocarbamate)
- (MGD)2–Fe–NO:
-
Mononitrosyl–iron complex
- Mo-enzymes:
-
Pterin–molybdenum-containing enzymes
- NaR:
-
Nitrate reductases
- NO:
-
Nitric oxide radical
- SO:
-
Sulfite oxidase
- XO:
-
Xanthine oxidase
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Acknowledgments
L.M. (SFRH/BPD/39036/2007) thanks Fundação para a Ciência e a Tecnologia, MCTES, for a fellowship grant. This work was supported by project PTDC/QUI-BIQ/100366/2008 financed by the Fundação para a Ciência e a Tecnologia, MCTES.
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Maia, L.B., Moura, J.J.G. Nitrite reduction by xanthine oxidase family enzymes: a new class of nitrite reductases. J Biol Inorg Chem 16, 443–460 (2011). https://doi.org/10.1007/s00775-010-0741-z
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DOI: https://doi.org/10.1007/s00775-010-0741-z