Flavodiiron proteins (FDPs) are a family of enzymes endowed with bona fide oxygen- and/or nitric-oxide reductase activity, although their substrate specificity determinants remain elusive. After a comprehensive comparison of available three-dimensional structures, particularly of FDPs with a clear preference toward either O2 or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Tyr(271) and Lys(53) in the oxygen reducing Entamoeba histolytica EhFdp1. The biochemical and biophysical properties of these mutants were studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies coupled to potentiometry. Their reactivity with O2 and NO was analyzed by stopped-flow absorption spectroscopy and amperometric methods. These mutations, whereas keeping the overall properties of the redox cofactors, resulted in increased NO reductase activity and faster inactivation of the enzyme in the reaction with O2,
Flavodiiron Oxygen Reductase from Entamoeba histolytica: MODULATION OF SUBSTRATE PREFERENCE BY TYROSINE 271 AND LYSINE 53 / V. L., Goncalves; J. B., Vicente; L., Pinto; C. V., Romao; C., Frazao; Sarti, Paolo; Giuffre', Alessandro; M., Teixeira. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - STAMPA. - 289:(2014), pp. 28260-28270. [10.1074/jbc.M114.579086]
Flavodiiron Oxygen Reductase from Entamoeba histolytica: MODULATION OF SUBSTRATE PREFERENCE BY TYROSINE 271 AND LYSINE 53
SARTI, Paolo;GIUFFRE', ALESSANDRO;
2014
Abstract
Flavodiiron proteins (FDPs) are a family of enzymes endowed with bona fide oxygen- and/or nitric-oxide reductase activity, although their substrate specificity determinants remain elusive. After a comprehensive comparison of available three-dimensional structures, particularly of FDPs with a clear preference toward either O2 or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Tyr(271) and Lys(53) in the oxygen reducing Entamoeba histolytica EhFdp1. The biochemical and biophysical properties of these mutants were studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies coupled to potentiometry. Their reactivity with O2 and NO was analyzed by stopped-flow absorption spectroscopy and amperometric methods. These mutations, whereas keeping the overall properties of the redox cofactors, resulted in increased NO reductase activity and faster inactivation of the enzyme in the reaction with O2,I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
