title: Probing the geometric and electronic structures of the low-temperature azide adduct and the product-inhibited form of oxidized manganese superoxide dismutase.
abstract: 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 redox-active 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-Mn3+ 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 H2O2. The implications of these results with respect to the mechanism of O2*- dismutation by MnSOD are discussed.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Jackson
authors.forename: Anush
authors.initials: A
authors.lastname: Karapetian
authors.forename: Anne-Frances
authors.initials: AF
authors.lastname: Miller
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Brunold
mesh_terms: D001386:Azides / Q000737:chemistry*; D001665:Binding Sites; D002942:Circular Dichroism; D003080:Cold Temperature; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004583:Electrons; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D008345:Manganese / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D010084:Oxidation-Reduction; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D013481:Superoxides / Q000737:chemistry; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology
chemical_list: D001386:Azides; D029968:Escherichia coli Proteins; D013481:Superoxides; D008345:Manganese; D013482:Superoxide Dismutase