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

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.