title: Superoxide reduction by a superoxide reductase lacking the highly conserved lysine residue.
abstract: Superoxide reductases (SORs) are the most recently identified superoxide detoxification systems, being found in microorganisms from the three domains of life. These enzymes are characterized by a catalytic mononuclear iron site, with one cysteine and four histidine ligands of the ferrous active form. A lysine residue in the -EKHVP- motif, located close to the active site, has been considered to be essential for the enzyme function, by contributing to the positive surface patch that attracts the superoxide anion and by controlling the chemistry of the catalytic mechanism through a hydrogen bond network. However, we show here that this residue is substituted by non-equivalent amino acids in several putative SORs from Archaea and unicellular Eukarya. In this work, we focus on mechanistic and spectroscopic studies of one of these less common enzymes, the SOR from the hyperthermophilic Crenarchaeon Ignicoccus hospitalis. We employ pulse radiolysis fast kinetics and spectroscopic approaches to study the wild-type enzyme (-E23T24HVP-), and two mutants, T24K and E23A, the later mimicking enzymes lacking both the lysine and glutamate (a ferric ion ligand) of the motif. The efficiency of the wild-type protein and mutants in reducing superoxide is comparable to other SORs, revealing the robustness of these enzymes to single mutations.
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal.|Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, 17177, Stockholm, Sweden.
authors.forename: Ana F
authors.initials: AF
authors.lastname: Pinto
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal.
authors.forename: Célia V
authors.initials: CV
authors.lastname: Romão
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal.
authors.forename: Liliana C
authors.initials: LC
authors.lastname: Pinto
authors.affiliation: Lehrstuhl fuer Mikrobiologie, Universität Regensburg, 93053, Regensburg, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Huber
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal.
authors.forename: Lígia M
authors.initials: LM
authors.lastname: Saraiva
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal.
authors.forename: Smilja
authors.initials: S
authors.lastname: Todorovic
authors.affiliation: Chemistry Department, Brookhaven National Laboratory, Upton, NY, 11973-5000, USA.
authors.forename: Diane
authors.initials: D
authors.lastname: Cabelli
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Apartado 127, 2781-901, Oeiras, Portugal. miguel@itqb.unl.pt.
authors.forename: Miguel
authors.initials: M
authors.lastname: Teixeira
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D017124:Conserved Sequence; D019620:Desulfurococcaceae / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D013481:Superoxides / Q000737:chemistry*
chemical_list: D019843:Archaeal Proteins; D013481:Superoxides; D010088:Oxidoreductases; C121849:superoxide reductase; D008239:Lysine