Structure and dynamics of copper-free SOD: The protein before binding copper

The solution structure of the copper-free state of a monomeric form of superoxide dismutase (153 amino acids) was determined through C-13 and N-15 labeling. The protein contained two mutations at the native subunit-subunit interface (F50E and G51E) to obtain a soluble monomeric species and a mutation in the active site channel (E133Q). About 93% of carbon atoms, 95% of nitrogen atoms, and 96% of the protons were assigned. A total of 2467 meaningful NOES and 170 dihedral angles provided a family of 35 conformers Lwith RMSD values of 0.76 +/- 0.09 Angstrom for the backbone and 1.22 +/- 0.13 Angstrom for all heavy atoms. The secondary structure elements, connected by loops, produce the typical superoxide dismutase Greek key fold, formed by an eight-stranded beta-barrel. The comparison with the copper-bound monomeric and dimeric structures shows that the metal ligands have a conformation very close to that of the copper-bound forms. This feature indicates that the copper-binding site is preorganized and well ordered also in the absence of the copper ion. The active-site channel shows a sizable increase in width, achieving a suitable conformation to receive the copper ion. The histidines ring NH resonances that bind the copper ion and the region around the active-site channel experience, as found from N-15 relaxation studies, conformational exchange processes. The increased width of the channel and the higher mobility of the histidine rings of the copper site in the copper-free form with respect to the holoprotein is discussed in terms of the process of copper insertion.