Anion binding properties of reduced and oxidized iron-containing superoxide dismutase reveal no requirement for tyrosine 34

Anne Frances Miller, David L. Sorkin, K. Padmakumar

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

We report the first spectroscopic observation of substrate analogue binding to the reduced state of iron superoxide dismutase from Escherichia coli (Fe2+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 KM of Fe2+SOD. Thus, it appears that substrate binding to Fe2+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 (Fe3+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 Fe3+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 N3- 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 Fe3+. 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.

Original languageEnglish
Pages (from-to)5969-5981
Number of pages13
JournalBiochemistry
Volume44
Issue number16
DOIs
StatePublished - Apr 26 2005

ASJC Scopus subject areas

  • Biochemistry

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