| The highly homologous Fe- and Mn-containing superoxide dismutases (FeSOD and MnSOD) from Escherichia coli nonetheless display striking metal specificities, therefore providing ideal systems through which to explore the role of the second sphere in tuning metalloenzyme active site properties. A combination of spectroscopic and computational methods have been utilized to study the geometric and electronic properties of the wildtype FeSOD and MnSOD, the catalytically inactive Fe-substituted MnSOD (Fe:MnSOD) and Mn-substituted FeSOD (Mn:FeSOD), and several variants of FeSOD with varying degrees of catalytic activity, including the Q69H, Q69A, and Q69E mutants.; Despite their dramatically different catalytic activities, species in the Fe-bound (FeSOD, Fe:MnSOD and the Q69X variants of FeSOD) and the Mn-bound proteins (MnSOD, Mn:FeSOD) respectively exhibit virtually identical absorption, circular dichroism (CD), magnetic CD (MCD) and VTVH MCD data. Therefore spectroscopy alone cannot provide direct insight into the mechanism by which catalytic activity is tuned by SODS. Through the use of computational methods, including density functional theory and semi-empirical INDO/S-CI calculations, different interactions between the second sphere and the active site metal in FeSOD and MnSOD have been identified and their significance with respect to catalytic activity has been explored.; Although subtle differences in the second sphere do not directly influence the active-site structures and the spectroscopic signatures of the resting enzymes, binding of azide (a substrate analogue) to the oxidized enzymes induces striking differences in both crystallographic and spectroscopic data. Most intriguingly, a remarkable correlation has been established between the energies of the dominant N3 → Fe3+ ligand-to-metal charge transfer transition and the reduction midpoint potentials of the resting enzymes as well as the strengths of the H-bond interaction between the second sphere and the coordinated solvent. Combined quantum mechanics/molecular mechanics methodologies in conjunction with INDO/S-CI calculations have been used to generate experimentally-validated active-site models for the azide complexes of FeSOD, Fe:MnSOD, and the Q69A, Q69E FeSOD mutants, and the geometric and electronic factors contributing to the distinct second-sphere tuning of active site/substrate analogue interactions have been explored. |
