Single crystal polarized spectroscopy of manganese superoxide dismutase and electronic structure of the active site metal complex

Manganese superoxide dismutase from E. coli crystallizes from poly(ethylene glycol) solution in the orthorhombic space group C222₁. The bladelike purple crystals of the Mn(III) enzyme are strongly pleiochroic in polarized light, appearing red or green for light polarized parallel or perpendicular to their long axis, coincident with the crystallographic c-axis. Polarized spectra from oriented single crystals of Mn superoxide dismutase reveal a dramatic dichroism of the optical absorption bands that is primarily associated with a single ligand field transition near 500 nm exhibiting a polarization ratio of greater than 10:1, with amplitude maximizing for polarization parallel to the crystallographic c-axis. Detailed analysis of this polarization by projection onto the four Mn sites within the asymmetric unit allows interpretation of the crystal spectra in terms of molecular excitations and active site electronic structure. The strongest polarization is roughly aligned with the carboxylate ligand, suggesting a significant component of carboxylate-to-Mn(III) ligand-to-metal charge-transfer (LMCT) character to this absorption band. Density functional theory calculations on the ground-state electronic structure of an active site model predict strong mixing between the valence levels of the Mn(III) ion and both hydroxide and carboxylate oxyanion donor groups. Furthermore, this indicates that optical polarization in the transition to the second electronic excited state arises from the directional covalency in these metal-ligand interactions. Calculations of geometric and electronic structures of the reduced and protonated Mn(II)(H₂O) model lead to the prediction that the Mn-O bond stretch coordinate is important for electron-transfer reactivity in the active site. The combination of experimental and computational approaches provides insight into the contributions of endogenous ligands to the electronic structure of the Mn(III) ground state for this important biological redox complex.