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Nuclear magnetic resonance (NMR) provides detailed insights into the conformational features of unfolded and partially folded proteins. In the case of metalloproteins, special attention should be devoted to the characterization of the properties of the metal binding sites, and specific approaches need to be developed depending on the nature of the metal ion and its coordination environment. At the same time, metal-based NMR parameters may help in getting a better picture of the average structural properties of the metalloprotein. A critical evaluation of the limits of applicability of paramagnetic effects for solution structure determination in partially folded or unfolded proteins is presented. The coupling between NMR characterization of structure and dynamic of the polypeptide chain and of the metal environment provides insights into the stabilizing role of metal ions in metalloproteins. The overall approach is illustrated for some case examples of increasing flexibility obtained far from native conditions for cytochrome c and superoxide dismutase, two metalloproteins that have been extensively studied in our lab and whose misfolded forms may be relevant for important biological processes.

The interaction of fluoride with copper zinc superoxide dismutase (SOD) has been reinvestigated by using both bovine and yeast isoenzymes. The affinity of anions for the latter isoenzyme is larger, and this avoids much of the ionic strength effects connected with weak binding ligands. 19 F NMR studies on fluoride in presence of yeast SOD confirm that the anion binds the copper ion. 1 H NMR studies of the Cu 2 Co 2 SOD derivatives in the presence of F − indicate that no ligand is removed from coordination

Substitution of zinc (II) with cobalt (II) in copper zinc superoxide dismutase gives rise to a derivative suitable for 1 H NMR investigation owing to the magnetic coupling between the two metal ions. 1 H NOE measurements have been performed in order to relate them to interproton distances