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Moura I, Maia LB, Pauleta SR, Moura JJG. "A bird’s-eye view of denitrification in relation to the nitrogen cycle." In: Moura I, Moura JJG, Pauleta SR, Maia LB, eds. Metalloenzymes in Denitrification: Applications and Environmental Impacts, RSC Metallobiology Series No. 9 (ISBN: 978-1-78262-376-2). Vol. 39. The Royal Society of Chemistry; 2017:. Abstract

The involvement of xanthine oxidase (XO) in some reactive oxygen species (ROS) -mediated diseases has been proposed as a result of the generation of O-2(.-) and H2O2 during hypoxanthine and xanthine oxidation. In this study, it was shown that purified rat liver XO and xanthine dehydrogenase (XD) catalyse the NADH oxidation, generating O-2(.-) and inducing the peroxidation of liposomes, in a NADH and enzyme concentration-dependent manner. Comparatively to equimolar concentrations of xanthine, a higher peroxidation extent is observed in the presence of NADH. In addition, the peroxidation extent induced by XD is higher than that observed with XO. The in vivo-predominant dehydrogenase is, therefore, intrinsically efficient at generating ROS, without requiring the conversion to XO. Our results suggest that, in those pathological conditions where an increase on NADH concentration occurs, the NADH oxidation catalysed by XD may constitute an important pathway for ROS-mediated tissue injuries.

Maia LB, Moura I, Moura JJG. "EPR spectroscopy on mononuclear molybdenum-containing enzymes." In: Hanson G, Berliner LJ, eds. Metalloenzymes/Metalloproteins EPR, Biological Magnetic Resonance Vol. 33. Vol. 12. Springer Publishers; 2017:. 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 O-2 (center dot-) 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 O-2 (center dot-) 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(center dot-), 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 mu M-1 min(-1)) was found to be higher than that of the XO specificity constant (1.07 +/- 0.09 mu M-1 min(-1)). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O-2 (center dot-) source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O-2 (center dot-) 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.

Maia LB, Moura JJG. "Lessons from denitrification for the human metabolism of signalling nitric oxide." In: Moura I, Moura JJG, Pauleta SR, Maia LB, eds. Metalloenzymes in Denitrification: Applications and Environmental Impacts, RSC Metallobiology Series No. 9 (ISBN: 978-1-78262-376-2). Vol. 41. The Royal Society of Chemistry; 2017:.
Maia LB, Moura I, Moura JJG. "Molybdenum and tungsten-containing enzymes: an overview." In: Hille R, Schulzke C, Kirk M, eds. Molybdenum and Tungsten Enzymes: Biochemistry, RSC Metallobiology Series No. 5 (ISBN: 978-1-78262-089-1). Vol. 28. The Royal Society of Chemistry; 2017:.
Maiti BK, Maia LB, Silveira CM, Todorovic S, Carreira C, Carepo MS, Grazina R, Moura I, Pauleta SR, Moura JJG. "Incorporation of molybdenum in rubredoxin: models for mononuclear molybdenum enzymes." Journal of Biological Inorganic Chemistry. 2015;20:821-829. AbstractWebsite

Molybdenum is found in the active site of enzymes usually coordinated by one or two pyranopterin molecules. Here, we mimic an enzyme with a mononuclear molybdenum-bis pyranopterin center by incorporating molybdenum in rubredoxin. In the molybdenum-substituted rubredoxin, the metal ion is coordinated by four sulfurs from conserved cysteine residues of the apo-rubredoxin and two other exogenous ligands, oxygen and thiol, forming a Mo((VI))-(S-Cys)4(O)(X) complex, where X represents -OH or -SR. The rubredoxin molybdenum center is stabilized in a Mo(VI) oxidation state, but can be reduced to Mo(IV) via Mo(V) by dithionite, being a suitable model for the spectroscopic properties of resting and reduced forms of molybdenum-bis pyranopterin-containing enzymes. Preliminary experiments indicate that the molybdenum site built in rubredoxin can promote oxo transfer reactions, as exemplified with the oxidation of arsenite to arsenate.

Moura JJG, Bernhardt PV, Maia LB, Gonzalez PJ. "Molybdenum and tungsten enzymes: from Biology to chemistry and back." Journal of Biological Inorganic Chemistry. 2015;20:181-182.Website
Maia LB, Moura JJG, Moura I. "Molybdenum and tungsten-dependent formate dehydrogenases." Journal of Biological Inorganic Chemistry. 2015;20:287-309. AbstractWebsite


Maia LB, Moura JJG. "Nitrite reduction by molybdoenzymes: a new class of nitric oxide-forming nitrite reductases." Journal of Biological Inorganic Chemistry. 2015;20:403-433. AbstractWebsite


Maia LB, Moura JJG. "How Biology handles nitrite." Chemical Reviews. 2014;114:5273-5357. AbstractWebsite


Moura JJG, Maiti BK, Carreira C, Maia LB, Carepo SP, Pauleta SR, Moura I. "Metal substituted rubredoxins: a sulfur rich coordination site as models for metalloenzymes." Journal of Biological Inorganic Chemistry. 2014;19:731. AbstractWebsite


Maiti BK, Maia LB, Pal K, Pakhira B, Aviles T, Moura I, Pauleta SR, Nunez JL, Rizzi AC, Brondino CD, Sarkar S, Moura JJG. "One electron reduced square planar bis(benzene-1,2-dithiolato) copper dianionic complex and redox switch by O2/HO-." Inorganic Chemistry. 2014;53:12799-12808. AbstractWebsite


Maia LB, Moura JJG. "Nitrite reduction by xanthine oxidase family enzymes: a new class of nitrite reductases." Journal of Biological Inorganic Chemistry. 2011;16:443-460. AbstractWebsite

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.