Emine Yikilmaz

Mutational and spectroscopic studies of the significance of the active site glutamine to metal ion specificity in superoxide dismutase

We are addressing the puzzling metal ion specificity of Fe- and Mn-containing superoxide dismutases (SODs) [see C.K.Vance, A.-F. Miller. J. Am. Chem. Soc. 120(3) (1998) 461-467]. Here, we test the significance to activity and active site integrity of the Gln side chain at the center of the active site hydrogen bond network. We have generated a mutant of MnSOD with the active site Gln in the location characteristic of Fe-specific SODs. The active site is similar to that of MnSOD when Mn2+, Fe3+ or Fe2+ are bound, based on EPR and NMR spectroscopy. However, the mutants Fe-supported activity is at least 7% that of FeSOD, in contrast to Fe(Mn)SOD, which has 0% of FeSODs activity. Thus, moving the active site Gln converts Mn-specific SOD into a cambialistic SOD and the Gln proves to be important but not the sole determinant of metal-ion specificity. Indeed, subtle differences in the spectra of Mn2+, Fe3+ and 1H in the presence of Fe2+ distinguish the G77Q, Q146A mut-(Mn)SOD from WT (Mn)SOD, and may prove to be correlated with metal ion activity. We have directly observed the side chain of the active site Gln in Fe2+ SOD and Fe2+ (Mn)SOD by 15N NMR. The very different chemical shifts indicate that the active site Gln interacts differently with Fe2+ in the two proteins. Since a shorter distance from Gln to Fe and stronger interaction with Fe correlate with a lower Em in Fe(Mn)SOD, Gln has the effect of destabilizing additional electron density on the metal ion. It may do this by stabilizing OH- coordinated to the metal ion.

Spectroscopic and Computational Investigation of Second-Sphere Contributions to Redox Tuning in Escherichia coli Iron Superoxide Dismutase

In Fe- and Mn-dependent superoxide dismutases (SODs), second-sphere residues have been implicated in precisely tuning the metal ion reduction potential to maximize catalytic activity (Vance, C. K.; Miller, A.-F. J. Am. Chem. Soc. 1998, 120, 461-467). In the present study, spectroscopic and computational methods were used to characterize three distinct Fe-bound SOD species that possess different second-coordination spheres and, consequently, Fe(3+/2+)reduction potentials that vary by approximately 1 V, namely, FeSOD, Fe-substituted MnSOD (Fe(Mn)SOD), and the Q69E FeSOD mutant. Despite having markedly different metal ion reduction potentials, FeSOD, Fe(Mn)SOD, and Q69E FeSOD exhibit virtually identical electronic absorption, circular dichroism, and magnetic circular dichroism (MCD) spectra in both their oxidized and reduced states. Likewise, variable-temperature, variable-field MCD data obtained for the oxidized and reduced species do not reveal any significant electronic, and thus geometric, variations within the Fe ligand environment. To gain insight into the mechanism of metal ion redox tuning, complete enzyme models for the oxidized and reduced states of all three Fe-bound SOD species were generated using combined quantum mechanics/molecular mechanics (QM/MM) geometry optimizations. Consistent with our spectroscopic data, density functional theory computations performed on the corresponding active-site models predict that the three SOD species share similar active-site electronic structures in both their oxidized and reduced states. By using the QM/MM-optimized active-site models in conjunction with the conductor-like screening model to calculate the proton-coupled Fe(3+/2+) reduction potentials, we found that different hydrogen-bonding interactions with the conserved second-sphere Gln (changed to Glu in Q69E FeSOD) greatly perturb the p K of the Fe-bound solvent ligand and, thus, drastically affect the proton-coupled metal ion reduction potential.

Spectroscopic and computational insights into second-sphere amino-acid tuning of substrate analogue/active-site interactions in iron(III) superoxide dismutase.

In this study, the mechanism by which second-sphere residues modulate the structural and electronic properties of substrate-analogue complexes of the Fe-dependent superoxide dismutase (FeSOD) has been explored. Both spectroscopic and computational methods were used to investigate the azide (N3(-)) adducts of Fe(3+)SOD (N3-Fe(3+)SOD) and its Q69E mutant, as well as Fe(3+)-substituted MnSOD (N3-Fe(3+)(Mn)SOD) and its Y34F mutant. Electronic absorption, circular dichroism, and magnetic circular dichroism spectroscopic data reveal that the energy of the dominant N3(-)-->Fe(3+) ligand-to-metal charge transfer (LMCT) transition decreases in the order N3-Fe(3+)(Mn)SOD>N3-Fe(3+)SOD>Q69E N3-Fe(3+)SOD. Intriguingly, the LMCT transition energies correlate almost linearly with the Fe(3+/2+) reduction potentials of the corresponding Fe(3+)-bound SOD species in the absence of azide, which span a range of approximately 1 V (see the preceding paper). To explore the origin of this correlation, combined quantum mechanics/molecular mechanics (QM/MM) geometry optimizations were performed on complete enzyme models. The INDO/S-CI computed electronic transition energies satisfactorily reproduce the experimental trend in LMCT transition energies, indicating that the QM/MM optimized active-site models are reasonable. Density functional theory calculations on these experimentally validated active-site models reveal that the differences in spectral and electronic properties among the four N 3(-) adducts arise primarily from differences in the hydrogen-bond network involving the conserved second-sphere Gln (mutated to Glu in Q69E FeSOD) and the solvent ligand. The implications of our findings with respect to the mechanism by which the second-coordination sphere modulates substrate-analogue binding as well as the catalytic properties of FeSOD are discussed.