title: Flavodiiron oxygen reductase from Entamoeba histolytica: modulation of substrate preference by tyrosine 271 and lysine 53.
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.
authors.affiliation: From the Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República (EAN), 2781-901 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.
authors.forename: Vera L
authors.initials: VL
authors.lastname: Gonçalves
authors.affiliation: 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, joaovicente@ff.ul.pt.
authors.forename: João B
authors.initials: JB
authors.lastname: Vicente
authors.affiliation: From the Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República (EAN), 2781-901 Oeiras, Portugal.
authors.forename: Liliana
authors.initials: L
authors.lastname: Pinto
authors.affiliation: From the Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República (EAN), 2781-901 Oeiras, Portugal.
authors.forename: Célia V
authors.initials: CV
authors.lastname: Romão
authors.affiliation: From the Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República (EAN), 2781-901 Oeiras, Portugal.
authors.forename: Carlos
authors.initials: C
authors.lastname: Frazão
authors.affiliation: the Department 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.
authors.forename: Paolo
authors.initials: P
authors.lastname: Sarti
authors.affiliation: the CNR Institute of Biology, Molecular Medicine and Nanobiotechnology, Piazzale Aldo Moro 5, I-00185 Rome, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Giuffrè
authors.affiliation: From the Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República (EAN), 2781-901 Oeiras, Portugal, miguel@itqb.unl.pt.
authors.forename: Miguel
authors.initials: M
authors.lastname: Teixeira
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004748:Entamoeba histolytica / Q000737:chemistry* / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009569:Nitric Oxide; D014443:Tyrosine; D007501:Iron; D010088:Oxidoreductases; C034747:nitric-oxide reductase; D008239:Lysine; D010100:Oxygen
keywords: Flavoprotein; Iron; Nitric Oxide; Nitrosative Stress; Oxidation-Reduction (Redox); Oxidative Stress; Oxygen