Mapping the effects of metal ion reduction and substrate analog binding to Fe-superoxide dismutase by NMR spectroscopy

Fe-containing Superoxide dismutase (FeSOD) is an excellent model for studies of the role of the protein in shaping redox catalytic activity in metalloenzymes. In order to use NMR spectroscopy to monitor the protons in FeSOD, we have assigned all the observable backbone resonances in the HN heteronuclear single quantum correlation spectrum (HN-HSQC), in both of the reduced and oxidized states. This task required ²H, ¹³C and ¹⁵N triple labeling and quadruple resonance techniques due to FeSOD's molecular weight of 42kDa and paramagnetic high-spin active site Fe. The unobserved and unassigned residues of FeSOD are accounted for by paramagnetic relaxation and slow back-exchange of solvent protons into backbone H^N positions. Of FeSOD's 192 residues, we have previously assigned resonances for 118 in the oxidized state and now 141 in the reduced state. For residues > 14 Å from Fe, resonances are observable in both oxidation states. Relatively small chemical shift changes were found to accompany Fe²⁺ oxidation, but these extend throughout the protein. Specific binding of the substrate analog F^- to Fe²⁺SOD was found to involve residues near Tyr34, consistent with participation of this residue in substrate binding. This represents the first location of substrate binding to Fe²⁺SOD. Additional residues at the dimer interface are sensitive to a variety of anions in both of FeSOD's oxidation states and also Fe oxidation, consistent with either interaction with substrate or domain movement associated with substrate binding and metal ion oxidation.