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(eth ylene glycol) solution in the orthorhombic space group C222(1). The bl adelike purple crystals of the Mn(III) enzyme are strongly pleiochroic in polarized light, appearing red or green for light polarized parall el or perpendicular to their long axis, coincident with the crystallog raphic c-axis. Polarized spectra from oriented single crystals of Mn s uperoxide dismutase reveal a dramatic dichroism of the optical absorpt ion bands that is primarily associated with a single ligand field tran sition near 500 nm exhibiting a polarization ratio of greater than 10: 1, with amplitude maximizing for polarization parallel to the crystall ographic c-axis. Detailed analysis of this polarization by projection onto the four Mn sites within the asymmetric unit allows interpretatio n of the crystal spectra in terms of molecular excitations and active site electronic structure. The strongest polarization is roughly align ed with the carboxylate ligand, suggesting a significant component of carboxylate-to-Mn(III) ligand-to-metal charge-transfer (LMCT) characte r to this absorption band. Density functional theory calculations on t he ground-state electronic structure of an active site model predict s trong mixing between the valence levels of the Mn(III) ion and both hy droxide and carboxylate oxyanion donor groups. Furthermore, this indic ates that optical polarization in the transition to the second electro nic excited state arises from the directional covalency in these metal -ligand interactions. Calculations of geometric and electronic structu res of the reduced and protonated Mn(II)(H2O) model lead to the predic tion that the Mn-O bond stretch coordinate is important for electron-t ransfer 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) groun d state for this important biological redox complex.