Copper, zinc superoxide dismutase is a dimeric enzyme, and it has been shown that no cooperativity between the two subunits of the dimer is operative. The substitution of two hydrophobic residues, Phe 50 and Gly 51, with two Glu's at the interface region has disrupted the quaternary structure of the protein, thus producing a soluble monomeric form. However, this monomeric form was found to have an activity lower than that of the native dimeric species (10%). To answer the fundamental question of the role of the quaternary structure in the catalytic process of superoxide dismutase, we have determined the solution structure of the reduced monomeric mutant through NMR spectroscopy. Another fundamental issue with respect to the enzymatic mechanism is the coordination of reduced copper, which is the active center. The three-dimensional solution structure of this 153-residue monomeric form of SOD (16 kDa) has been determined using distance and dihedral angle constraints obtained from 13C, 15N triple-resonance NMR experiments. The solution structure is represented by a family of 36 structures, with a backbone rmsd of 0.81 +/- 0.13 A over residues 3-150 and of 0.56 +/- 0.08 A over residues 3-49 and 70-150. This structure has been compared with the available X-ray structures of reduced SODs as well as with the oxidized form of human and bovine isoenzymes. The structure contains the classical eight-stranded Greek key beta-barrel. In general, the backbone and the metal sites are not affected much by the monomerization, except in the region involved in the subunit-subunit interface in the dimeric protein, where a large disorder is present. Significative changes are observed in the conformation of the electrostatic loop, which forms one side of the active site channel and which is fundamental in determining the optimal electrostatic potential for driving the superoxide anions to the copper site which is the rate-limiting step of the enymatic reaction under nonsaturating conditions. In the present monomer, its conformation is less favorable for the diffusion of the substrate to the reaction site. The structure of the copper center is well-defined; copper(I) is coordinated to three histidines, at variance with copper(II) which is bound to four histidines. The hydrogen atom which binds the histidine nitrogen detached from copper(I) is structurally identified.

An extensive series of three‐dimensional, triple resonance experiments were performed on a fully labeled 13C, 15N monomeric form of human copper/zinc superoxide dismutase which contains 153 amino acids. The present system, in addition to the mutations at the subunit–subunit interface (Phe50→Glu, Gly51→Glu) which lead to the monomeric form, carries the mutation of the Glu133 to Gln, a residue at the active channel entrance. Firm assignment was found for 97% of the protons, 99% of the 13Cα, 91% of the 15N, 98% of 13C(O) and 90% of the 13Cβ backbone resonances. Analysis of the chemical shift values of 13Cα, Hα, 13Cβ and 13CO, of 3JHNHα coupling constants, of NOEs between backbone protons and of the H–D exchange behavior of amide protons permits the unequivocal detection of elements of secondary structure. The secondary structure of the present monomeric form is very similar to that of the WT enzyme, although the enzymatic activity is 20% of that of the WT protein. © 1997 John Wiley & Sons, Ltd.