Exploring the mechanisms of redox potential tuning and substrate/active-site interactions in iron- and manganese-bound superoxide dismutases and synthetic analogues

Iron- and manganese superoxide dismutases (Fe- and MnSODs) prevent the accumulation of superoxide ( O•-2 ) by using a redox active metal center to catalyze the disproportionation of two equivalents of O•-2 and two protons to O2 and H2O2. To probe the mechanisms of redox potential (Em) tuning and the nature of the substrate/active-site interactions in these enzymes and synthetic small-molecule analogues, we have used electronic absorption, circular dichroism (CD), magnetic CD, resonance Raman, and electron paramagnetic resonance spectroscopies. These experimental studies were complemented by density functional theory (DFT) and time-dependent DFT calculations to generate experimentally validated geometric and electronic structure descriptions for these species in the absence and in the presence of azide, an analogue of O•-2 .;Our experimentally validated computational models of wild-type (WT) MnSOD and Mn-substituted FeSOD (Mn(Fe)SOD) show only slight differences in the first coordination sphere; however, a second-sphere glutamine (Gln) residue is distinctly positioned in each of the two enzymes. Based on the excellent agreement between our computationally predicted Ems and those observed experimentally, we conclude that the differing positions of the Gln residue in the two protein matrices is the primary reason for the vastly different Ems observed for MnSOD and Mn(Fe)SOD. Similar studies of WT FeSOD and its Gln69 to His variant (Q69H FeSOD) have revealed that the elimination of constraining hydrogen bonds by the protein matrix results in a high degree of conformational freedom for the His residue (relative to the Gln residue in WT FeSOD), leading to an elevated Em and a decrease in the activity of this variant.;From a similar study of a functional mimic of FeSOD, Fe(dapsox), we conclude that a combination of proton uptake accompanying metal ion reduction and metal-ligand π-back-bonding in the reduced state serves to properly tune Em of this complex for SOD catalysis. Additionally, structural, spectroscopic, and computational studies of the azide adduct of Fe(dapsox) show that the azide/FeIII(dapsox) interactions are quite similar to those observed for the azide adduct of FeIIISOD, consistent with the high SOD activity of Fe(dapsox) and supporting an inner-sphere mechanism for catalysis.