The solution structure of the demetalated copper, zinc superoxide dismutase is obtained for the monomeric Glu133Gln/Phe50Glu/Gly51Glu mutant through NMR spectroscopy. The demetalated protein still has a well-defined tertiary structure; however, two beta-strands containing two copper ligands (His46 and His48, beta4) and one zinc ligand (Asp83, beta5) are shortened, and the sheet formed by these strands and strands beta7 and beta8 moves away from the other strands of the beta-barrel to form an open clam with respect to a closed conformation in the holoprotein. Furthermore, loop IV which contains three zinc ligands (His63, His71, and His80) and loop VII which contributes to the definition of the active cavity channel are severely disordered, and experience extensive mobility as it results from thorough (15)N relaxation measurements. These structural and mobility data, if compared with those of the copper-depleted protein and holoprotein, point out the role of each metal ion in the protein folding, leading to the final tertiary structure of the holoprotein, and provide hints for the mechanisms of metal delivery by metal chaperones.

The backbone assignment of reduced human dimeric Cu,Zn superoxide dismutase (SOD) was performed on a sample 100% enriched in (15)N, (13)C and 70% enriched in (2)H. (15)N T(1), T(2), and T(1)(rho) and (15)N-(1)H NOE assignment was performed at 600 MHz proton frequency on both wild-type SOD and the monomeric F50E/G51E/E133Q mutant. This allowed a comparison of the mobility in the subnanosecond and in the millisecond to microsecond time scales of the two systems. Both proteins are rather rigid, although some breathing of the beta sheets is detected in the wild type dimer. The monomer displays large mobility in the loops in the first part of the sequence, in loop IVa where point mutations have been introduced and at the C-terminus. The dimeric wild type is rigidified at loop IVa and at the C-terminus. Only loop VII shows a higher mobility in the dimer (besides some individual NH moieties). Conformational equilibria are displayed in the monomeric form around cysteines 57 and 146, thus explaining the disorder of arginine 143 which is the most important residue in guiding O(2)(-) toward the copper ion. The larger mobility in the wild type form with respect to the monomer in the picosecond to nanosecond time scale of helix alpha1 and loop VIIb, which provides the correct electrostatic driving force for O(2)(-) in the active channel, has been discussed in terms of favoring the activity of SOD.

The solution structure of homodimeric Cu 2 Zn 2 superoxide dismutase (SOD) of 306 aminoacids was determined on a 13 C, 15 N and 70% 2 H labeled sample. Two-thousand eighthundred and five meaningful NOEs were used, of which 96 intersubunit, and 115 dihedral angles provided a family of 30 conformers with an rmsd from the average of 0.78 ± 0.11 and 1.15 ± 0.09 Å for the backbone and heavy atoms, respectively. When the rmsd is calculated for each subunit, the values drop to 0.65 ± 0.09 and 1.08 ± 0.11 Å for the backbone and heavy atoms, respectively.The two subunits are identical on the NMR time scale, at variance with the X-ray structures that show structural differences between the two subunits as well as between different molecules in the unit cell. The elements of secondary structure, i.e. eight b sheets, are the same as in the X-ray structures and are well defined. The odd loops (I, III and V) are well resolved as well as loop II located at the subunit interface. On the contrary, loops IV and VI show some disorder. The residues of the active cavity are well defined whereas within the various subunits of the X-ray structure some are disordered or display different orientation in different X-ray structure determinations. The copper(I) ion and its ligands are well defined. This structure thus represents a well defined model in solution relevant for structure-function analysis of the protein. The comparison between the solution structure of monomeric mutants and the present structure shows that the subunit-subunit interactions increase the order in loop II. This has the consequences of inducing the structural and dynamic properties that are optimal for the enzymatic function of the wild-type enzyme. The regions 37-43 and 89-95, constituting loops III and V and the initial part of the b barrel and showing several mutations in familial amyotrophis lateral sclerosis (FALS)-related proteins have a quite extensive network of H-bonds that may account for their low mobility. Finally, the conformation of the key Arg143 residue is compared to that in the other dimeric and monomeric structures as well as in the recently reported structure of the CCS-superoxide dismutase (SOD) complex.