title: Anion binding properties of reduced and oxidized iron-containing superoxide dismutase reveal no requirement for tyrosine 34.
abstract: We report the first spectroscopic observation of substrate analogue binding to the reduced state of iron superoxide dismutase from Escherichia coli (Fe(2+)SOD) and demonstrate that the pH dependence reflects inhibition of anion binding by ionized Tyr34, not loss of a positive contribution on the part of Tyr34's labile proton. This can also explain the pH dependence of the K(M) of Fe(2+)SOD. Thus, it appears that substrate binding to Fe(2+)SOD occurs in the second sphere and is not strongly coupled to hydrogen bond donation. Parallel investigations of substrate analogue binding to the oxidized state (Fe(3+)SOD) confirm formation of a six-coordinate complex and resolve the apparent conflict with earlier nuclear magnetic relaxation dispersion (NMRD) results. Thus, we propose that two F(-) ions can bind to the oxidized Fe(3+)SOD active site, either displacing the coordinated solvent or lowering its exchange rate with bulk solvent. We show that neutral Tyr34's unfavorable effect on binding of the substrate analogue N(3)(-) can be ascribed to steric interference, as it does not apply to the smaller substrate analogues F(-) and OH(-). Finally, we report the first demonstration that HS(-) can act as a substrate analogue with regard both to redox reactivity with FeSOD and to ability to coordinate to the active site Fe(3+). Indeed, it forms a novel green complex. Thus, we have begun to evaluate the relative importance of different contributions that Tyr34 may make to substrate binding, and we have identified a novel, redox active substrate analogue that offers new possibilities for elucidating the mechanism of FeSOD.
authors.affiliation: Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218, USA. afm@uky.edu
authors.forename: Anne-Frances
authors.initials: AF
authors.lastname: Miller
authors.forename: David L
authors.initials: DL
authors.lastname: Sorkin
authors.forename: K
authors.initials: K
authors.lastname: Padmakumar
mesh_terms: D019943:Amino Acid Substitution; D000838:Anions / Q000378:metabolism; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013379:Substrate Specificity; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000838:Anions; D014443:Tyrosine; D007501:Iron; D013482:Superoxide Dismutase