Probing the geometric and electronic structures of the low-temperature azide adduct and the product-inhibited form of oxidized manganese superoxide dismutase

The geometric and electronic structures of the six-coordinate azide adduct of oxidized manganese superoxide dismutase (Mn³⁺SOD) that is formed at low temperatures, LT N₃-Mn³⁺SOD, has been examined in detail through a combined spectroscopic/computational approach. Electronic absorption, circular dichroism (CD), magnetic CD (MCD) and variable-temperature, variable-field (VTVH) MCD spectroscopies were used to determine electronic transition energies and to obtain an estimate of zero-field splitting parameters for LT N₃-Mn³⁺SOD. These experimental data were utilized in conjunction with semiempirical intermediate neglect of differential overlap/spectroscopic parametrization-configuration interaction (INDO/ S-CI) and time-dependent density functional theory (TD-DFT) computations to evaluate hypothetical active-site models of LT N₃-Mn³⁺SOD generated by constrained DFT geometry optimizations. Collectively, our spectroscopic/computational results indicate that N₃^- binding to Mn³⁺SOD at low temperatures promotes neither protonation of the axial solvent ligand nor reorientation of the redoxactive molecular orbital, both of which had been previously suggested. Using the same experimentally validated computational approach, models of the product-inhibited form of MnSOD were also developed and evaluated by their relative energies and TD-DFT-computed absorption spectra. On the basis of our computational results as well as previously published kinetic data, we propose that the product-inhibited form of MnSOD is best described as a side-on peroxo-Mn ³⁺ adduct possessing an axial H₂O ligand. Notably, attempts to generate a stable hydroperoxo-Mn³⁺SOD species by protonation of the proximal O atom of the hydroperoxo ligand resulted in dissociation of HOO^- and eventual H⁺ transfer from Tyr34 to HOO^-, generating deprotonated Tyr34 and H₂O ₂. The implications of these results with respect to the mechanism of O₂^.- dismutation by MnSOD are discussed.