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.

Proton-coupled electron transfer in Fe-superoxide dismutase and Mn-superoxide dismutase.

Fe-containing superoxide dismutase (FeSOD) and MnSOD are widely assumed to employ the same catalytic mechanism. However this has not been completely tested. In 1985, Bull and Fee showed that FeSOD took up a proton upon reduction [J. Am. Chem. Soc. 107 (1985) 3295]. We now demonstrate that MnSOD incorporates the same crucial coupling between electron transfer and proton transfer. The redox-coupled H(+) acceptor has been presumed to be the coordinated solvent molecule, in both FeSOD and MnSOD, however this is very difficult to test experimentally. We have now examined the most plausible alternative: that Tyr34 accepts a proton upon SOD reduction. We report specific incorporation of 13C in the C(zeta) positions of Tyr residues, assignment of the C(zeta) signal of Tyr34 in each of oxidized FeSOD and MnSOD, and direct NMR observations showing that in both cases, Tyr34 is in the neutral protonated state. Thus Tyr34 cannot accept a proton upon SOD reduction, and coordinated solvent is concluded to be the redox-coupled H(+) acceptor instead, in both FeSOD and MnSOD. We have also confirmed by direct 13C observation that the pK of 8.5 of reduced FeSOD corresponds to deprotonation of Tyr34. This work thus provides experimental proof of important commonalities between the detailed mechanisms of FeSOD and MnSOD.