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 Fe³⁺SOD (N₃-Fe³⁺SOD) and its Q69E mutant, as well as Fe³⁺-substituted MnSOD (N ₃-Fe³⁺(Mn)SOD) and its Y34F mutant. Electronic absorption, circular dichroism, and magnetic circular dichroism spectroscopic data reveal that the energy of the dominant N₃^- → Fe³⁺ ligand-to-metal charge transfer (LMCT) transition decreases in the order N ₃-Fe³⁺(Mn)SOD > N₃-Fe³⁺SOD > Q69E N₃-Fe³⁺SOD. Intriguingly, the LMCT transition energies correlate almost linearly with the Fe^3+/2+ reduction potentials of the corresponding Fe³⁺-bound SOD species in the absence of azide, which span a range of ∼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-Cl 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.