The solution structure of a monomeric, reduced form of human copper, zinc superoxide dismutase bearing the same charge as the native protein

The solution structure of a mutated (Phe50Glu, Gly51Glu, Val148Lys, Ile151L ys), reduced, monomeric form of human copper,zinc superoxide dismutase (SOD ; 153 amino acids) has been determined through 2237 meaningful nuclear Over hauser enhancements, out of 2492, and 43 dihedral angle constraints. A char acteristic of this mutant is that of having the same overall charge as the dimeric protein, but an activity of only 20% with respect to wild-type SOD. This protein, at a variance with a previously characterized monomeric form (Phe50Glu, Gly51Glu, Glu133Gln), does not contain mutations in the active site. Therefore, its characterization allows us to understand the structura l changes independently induced by the monomerization and by the active sit e mutation. The family of 36 conformers, which have a target funtion with r espect to the experimental constraints lower than 1.5 Angstrom(2), has RMSD values with respect to the average structure of 0.94+/-0.14 Angstrom(2) an d 1.50 +/- 0.14 Angstrom(2) for the backbone and the heavy atoms, respectiv ely. The overall folding, which includes the classical eight-stranded Greek -key beta-barrel and a short alpha-helix, is very close to that of the prev iously characterized monomeric mutant E133QM2SOD and to that of wild-type S OD. The region involved in the subunit-subunit interactions in the dimeric protein is confirmed to be disordered in the monomeric species. It is also observed that a sizable rearrangement of the charged groups of the electros tatic loop and of Arg143 takes place in the monomeric species. The width of the active site channel, both at its entrance and at the bottleneck of the active site, is discussed in the light of the influence on the enzymatic a ctivity and the latter with respect to the overall charge. It is also confi rmed that the NH proton of His63 shields the Cu(I) from the bulk solvent, t hus supporting the suggestion that superoxide may interact with the reduced metal ion in an outer-sphere fashion.