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.

The Crucial Importance of Chemistry in the Structure−Function Link: Manipulating Hydrogen Bonding in Iron-Containing Superoxide Dismutase,

“…Moreover, Tyr34 is responsible for the reduced state active site pK near 8.5 and exerts important control over substrate specificity (17,19,(39)(40)(41). In both mutants, Fe coordination geometry almost identical to that of the WT-FeSOD is retained (below).…”

Role of Conserved Tyrosine Residues in NiSOD Catalysis: A Case of Convergent Evolution

“…When azide is added to Y34F-Mn 3+ -SOD the enzyme resembles the low temperature six-coordinate N 3 -Mn 3+ -SOD complex, and shows no temperature dependence(71). Thus, the “gateway” tyrosine seems to play a critical role in controlling anion access to the metal site in both Fe- and Mn-SODs(15, 16, 71) by inhibiting anion binding at the metal center. The same function for Y9 appears to be a feature of NiSOD catalysis.…”

The structural biochemistry of the superoxide dismutases

“…In this mechanism the inactive inhibitor-bound enzyme is six-coordinated, while the active form remains five-coordinated, with substrate binding causing the displacement of one of the manganese ligands. A third proposed mechanism suggested that the catalytic reaction occurs through an outer sphere mechanism and instead utilizes an alternate anion-binding site in the active site, possibly at the base of the funnel [216–218]. This proposal is based upon various anions inhibiting the activity of FeSOD, but without directly coordinating the metal center.…”