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.