How Can a Single Second Sphere Amino Acid Substitution Cause Reduction Midpoint Potential Changes of Hundreds of Millivolts? Emine Yikilmaz, ²,‡ Jason Porta, § Laurie E. Grove, | Ardeschir Vahedi-Faridi, §,# Yuriy Bronshteyn, ² Thomas C. Brunold, | Gloria E. O. Borgstahl, § and Anne-Frances Miller* ,²,‡ Contribution from the Department of Chemistry, UniVersity of Kentucky, Lexington, Kentucky 40506-0055, Department of Chemistry, the Johns Hopkins UniVersity, Baltimore, Maryland 21218, Eppley Institute for Research in Cancer and Allied Diseases, 987696 Nebraska Medical Center, Omaha, Nebraska 68198-7696, and Department of Chemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706 Received December 22, 2006; E-mail: afm@uky.edu Abstract: The active site metal ion of superoxide dismutase (SOD) is reduced and reoxidized as it disproportionates superoxide to dioxygen and hydrogen peroxide. Thus, the reduction midpoint potential (Em) is a critical determinant of catalytic activity. In E. coli Fe-containing SOD (FeSOD), reduction of Fe 3+ is accompanied by protonation of a coordinated OH - , to produce Fe 2+ coordinated by H2O. The coordinated solvent’s only contact with the protein beyond the active site is a conserved Gln residue. Mutation of this Gln to His or Glu resulted in elevation of the Em by 220 mV and more than 660 mV, respectively [Yikilmaz et al., Biochemistry 2006, 45, 1151-1161], despite the fact that overall protein structure was preserved, His is a chemically conservative replacement for Gln, and neutral Glu is isostructural and isoelectronic with Gln. Therefore, we have investigated several possible bases for the elevated Em’s, including altered Fe electronic structure, altered active site electrostatics, altered H-bonding and altered redox-coupled proton transfer. Using EPR, MCD, and NMR spectroscopies, we find that the active site electronic structures of the two mutants resemble that of the WT enzyme, for both oxidation states, and Q69E-FeSOD’s apparent deviation from WT-like Fe 3+ coordination in the oxidized state can be explained by increased affinity for a small anion. Spontaneous coordination of an exogenous anion can only stabilize oxidized Q69E-Fe 3+ SOD and, therefore, cannot account for the increased Em of Q69E FeSOD. WT-like anion binding affinities and active site pK’s indicate that His69 of Q69H-FeSOD is neutral in both oxidation states, like Gln69 of WT- FeSOD, whereas Glu69 appears to be neutral in the oxidized state but ionized in the reduced state of Q69E-FeSOD. A 1.1 Å resolution crystal structure of Q69E-Fe 2+ SOD indicates that Glu69 accepts a strong H-bond from coordinated solvent in the reduced state, in contrast to the case in WT-FeSOD where Gln69 donates an H-bond. These data and DFT calculations lead to the proposal that the elevated Em of Q69E- FeSOD can be substantially explained by (1) relief from enforced H-bond donation in the reduced state, (2) Glu69’s capacity to provide a proton for proton-coupled Fe 3+ reduction, and (3) strong hydrogen bond acceptance in the reduced state, which stabilizes coordinated H2O. Our results thus support the hypothesis that the protein matrix can apply significant redox tuning via its influence over redox-coupled proton transfer and the energy associated with it. Introduction Superoxide dismutases (SODs) catalyze the disproportionation of 2 O 2 •- + 2H + to O 2 + H 2 O 2 . 1-3 The FeSODs and MnSODs are highly homologous at all levels of structure 4-6 but differ from the two other families of SOD, the Cu,ZnSODs, and the NiSODs. FeSODs and MnSODs are dimers or tetramers of ∼22 kDa monomers, each with its own active site. 7 Each active site contains a single Fe or Mn ion coordinated with distorted ² University of Kentucky. ‡ Johns Hopkins University. § Eppley Institute for Research in Cancer and Allied Diseases. | University of Wisconsin. # Current address: Department of Biochemistry/Crystallography, Free University Berlin, Takustr. 6, 14195 Berlin, Germany. (1) Lavelle, F.; McAdam, M. E.; Fielden, E. M.; Roberts, P. B.; Puget, K.; Michelson, A. M. Biochem. J. 1977, 161,3-11. (2) Bull, C.; Fee, J. A. J. Am. Chem. Soc. 1985, 107, 3295-3304. (3) McCord, J. M.; Fridovich, I. J. Biol. Chem. 1969, 244, 6049-6055. (4) Stallings, W. C.; Pattridge, K. A.; Strong, R. K.; Ludwig, M. L. J. Biol. Chem. 1984, 259 (17), 10695-10699. (5) Ringe, D.; Petsko, G. A.; Yamakura, F.; Suzuki, K.; Ohmori, D. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 3879-3883. (6) Lah, M. S.; Dixon, M. M.; Pattridge, K. A.; Stallings, W. C.; Fee, J. 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