Flavodiiron oxygen reductase from Entamoeba histolytica: Modulation of substrate preference by tyrosine 271 and lysine 53

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 O₂or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Tyr²⁷¹and Lys⁵³in the oxygen reducing Entamoeba histolytica EhFdp1. The biochemical and biophysical properties of these mutants were studied by UVvisible and electron paramagnetic resonance (EPR) spectroscopies coupled to potentiometry. Their reactivity with O₂and 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, pointing to a role of the mutated residues in substrate selectivity.