Assignment of the backbone resonances of oxidized Fe-superoxide dismutase, a 42 kDa paramagnet-containing enzyme.

Superoxide dismutases (SODs) disproportionate 2O·− 2 + 2H+ → H2O2 + O2, thus helping to forestall aging and protect aerobic organisms against the byproducts of respiration (reviewed in Miller and Sorkin, 1997). FeSOD is a dimer of identical 21 kDa monomers, each containing a single high-spin non-heme non-sulfur Fe in the active site. This Fe alternates between the Fe3+ and Fe2+ states in the accepted catalytic cycle (Miller and Sorkin, 1997), but is always high spin and thus paramagnetic. Proton transfer is believed to be the rate-limiting step of FeSOD catalysis (Bull and Fee, 1985), and hydrogen bond networks are proposed to support the active site and connect it to solvent. Thus, we wish to directly observe hydrogen bonding protons in FeSOD. We have begun by assigning the backbone 1HN, 15N, 13C′, 13Cα and 13Cβ resonances of perdeuterated Fe3+SOD suspended in H2O solution. We have obtained assignments for 61% of FeSOD’s 192 residues per monomer, which represents all observable backbone NH resonances. This is a significant achievement considering FeSOD’s size and the severe paramagnetic relaxivity of the active site high-spin Fe3+, and constitutes the first substantial backbone assignment of a member of the growing class of non-heme, non-sulfur Fe enzymes.