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.
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.