| Iron- and manganese superoxide dismutases (Fe- and MnSODs, respectively) display remarkable metal-ion specificities despite their nearly identical active-site structures. This specificity has been proposed to stem from second-sphere tuning of metal ion properties through a conserved hydrogen-bond network. To verify this proposal, a combination of spectroscopic and computational techniques were employed, including electronic absorption, circular dichroism, magnetic circular dichroism, and resonance Raman spectroscopies, as well as quantum mechanics/molecular mechanics (QM/MM) and density functional theory (DFT) calculations.;To elucidate the role of the conserved hydrogen-bond network, four SOD species were studied, FeSOD, Fe-substituted MnSOD (Fe(Mn)SOD), Q69E and Q69H FeSOD. Each species possesses a different hydrogen-bond interaction between the second-sphere Gln (or Glu or His) residue and solvent ligand, resulting in Fe reduction potentials that vary by ∼1 V and thus drastically reduced catalytic activity for the non-native species. Our spectroscopic data revealed minimal differences in the metal ion electronic structures. Alternatively, for FeSOD, Fe(Mn)SOD and Q69E FeSOD, computed proton-coupled reduction potentials based on QM/MM-generated active-site models indicated that different hydrogen-bonding interactions with the second-sphere Gin (Glu in Q69E FeSOD) greatly perturb the pK of the Fe-bound solvent ligand, thus drastically affecting the proton-coupled metal ion reduction potential. In contrast, disruption of this hydrogen-bond network, as occurs in the Q69H FeSOD mutant, resulted in a metal ion reduction potential that is minimally influenced by second sphere amino acids.;Furthermore, our spectroscopic data of the azide-adducts of Fe 3+SOD, Fe3+(Mn)SOD and Q69E Fe3+SOD, which serve as models of the substrate-bound state, revealed significant differences in the metal ion electronic structures, in sharp contrast to the results obtained for the resting states of these species. Collectively, these results suggest that the residues involved in the active-site hydrogen-bond network respond to azide coordination to the Fe3+ center in different ways, as dictated by the protein matrix, thereby leading to distinct electronic structures of the three azide complexes. |
