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 (NO adducts of Fe3+SOD (N-3-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 N-3(-) -> Fe3+ ligand-to-metal charge transfer (LMCT) transition decreases in the order N3-Fe3+(Mn)SOD > N-3-Fe3+SOD > Q69E N-3-Fe3+SOD. Intriguingly, the LMCT transition energies correlate almost linearly with the Fe3+/2+ reduction potentials of the corresponding Fe3+-bound SOD species in the absence of azide, which span a range of similar to 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 Gin (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.