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 towards either O2 or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Y271 and K53 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 ... More
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 towards either O2 or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Y271 and K53 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, while 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, pointing to a role of the mutated residues in substrate selectivity.
