The Fe-containing superoxide dismutases (Fe-SODs) catalyze conversion of superoxide to dioxygen and hydroge peroxide, thus forestalling aging and degenerative diseases. SOD's catalytic activity rests on its ability to provide protons and Eo between those of reduction and oxidation of superoxide ion. We propose NMR experiments to identify residues involved in proton transfer and redox tuning via electrostatic interactions and the active site hydrogen bond network. Comparison of the pKs of Tyr 34 in reduced and oxidized SOD, with and without substrate analogs bound will reveal whether Tyr 34 donates a proton to substrate upon Fe oxidation or upon binding. If the pK does not drop upon oxidation then coordinated solvent instead of Tyr 34 will be identified as the proton donor in that step. The difference between the pKs of the active site ionizable amino acids Tyr 34, His 30 and Tyr 76 in the two oxidation states will reveal the extent to which the protonation state of any of these are coupled to Fe's oxidation state. Thus we will elucidate coupling of proton transfer to substrate binding and electron transfer. Hydrogen bonding networks in the active site exert an important effect on both the thermodynamic and kinetic capabilities of the active site. The proposed work will identify protons in hydrogen bonds related to electron transfer, substrate binding and proton transfer (and thus catalytic activity) by functional H/D labeling. Replacement of a Gln residue central to the active site hydrogen bond network with a His will allow us to distinguish between structural perturbation of the active site (upon replacement of Gln with it's hydrogen bonding mimic neutral His), and disruption of hydrogen bonding upon subsequent protonation of His. Comparison of the exchange rates will identify hydrogen bonds affected by a change in the hydrogen bonding functionality of residue 69, and thus the active site hydrogen bond network. Thus we will elucidate coupling of proton transfer to electron transfer and probe the nature and significance of hydrogen bond networks. Both are ubiquitous, oft-proposed but poorly understood features of enzyme catalysis. NMR's ability to directly observe protons suits it ideally to the problem.
