The superoxide reductase from the early diverging eukaryote Giardia intestinalis |
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Authors: | Testa Fabrizio Mastronicola Daniela Cabelli Diane E Bordi Eugenio Pucillo Leopoldo P Sarti Paolo Saraiva Lígia M Giuffrè Alessandro Teixeira Miguel |
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Affiliation: | a Department of Biochemical Sciences, CNR Institute of Molecular Biology and Pathology, and Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, I-00185 Rome, Italyb Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973-5000, USAc Clinical Chemistry and Microbiology Laboratory, Istituto Nazionale per le Malattie Infettive IRCCS “Lazzaro Spallanzani,” Rome, Italyd Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal |
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Abstract: | Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O2•−) not through its dismutation, but via reduction to hydrogen peroxide (H2O2) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SORGi) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (Tfinal) with Fe3+ ligated to glutamate or hydroxide depending on pH (apparent pKa = 8.7). Although showing negligible SOD activity, reduced SORGi reacts with O2•− with a pH-independent second-order rate constant k1 = 1.0 × 109 M− 1 s− 1 and yields the ferric-(hydro)peroxo intermediate T1; this in turn rapidly decays to the Tfinal state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SORGi is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection. |
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Keywords: | ROS, reactive oxygen species SOR, superoxide reductase SOD, superoxide dismutase SORGi, superoxide reductase from Giardia intestinalis 1Fe-SOR, one-iron-containing SOR 2Fe-SOR, two-iron-containing SOR SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis MES, 2-(N-morpholino)ethanesulfonic acid HEPES, N-(2-hydroxyethyl)piperazine-N&prime -2-ethanesulfonic acid Tris, tris(hydroxymethyl)aminomethane SVD, singular value decomposition EDTA, ethylenediaminetetraacetate |
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