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, pointing to a role of the mutated residues in substrate selectivity.
Highlights
Flavodiiron proteins (FDPs) are O2 and/or nitric oxide (NO) reductases
After a comprehensive comparison of available three-dimensional structures, 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 Tyr271 and Lys53 in the oxygen reducing Entamoeba histolytica EhFdp1
By comparing the structures of two well characterized enzymes, we identified two residues in close spatial proximity to the diiron site that differ between the two enzymes: a tyrosine and a lysine in the protozoan FDP replaced by serine and aspartate, respectively, in the bacterial enzyme
Summary
Romão‡, Carlos Frazão‡, Paolo Sartiʈ**, Alessandro Giuffrè**, and Miguel Teixeira‡3 From the ‡Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República (EAN), 2781901 Oeiras, Portugal, the §Metabolism and Genetics Group, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal, the ¶Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal, the ʈDepartment of Biochemical Sciences and Istituto Pasteur, Fondazione Cenci Bolognetti, Sapienza University of Rome, Piazzale Aldo Moro 5, I-00185 Rome, Italy, and the **CNR Institute of Biology, Molecular Medicine and Nanobiotechnology, Piazzale Aldo Moro 5, I-00185 Rome, Italy
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