Newer Biophysical Investigations

The large number of histidine residues associated with the active site of bovine SOD facilitates the study of its chemical and structural properties by nuclear magnetic resonance (NMR) spectroscopy. Mainly the downfield histidine imidazole N-proton resonances were subject of a large quantity of investigations The deuteration of histidine residues is dependent only on the absence of co-ordinated metal ions and not changes in the protein structure Therefore, in apo-, zinc- and holo-super-oxide dismutase, only those histidine residues not co-ordinated to metal ions will be deuterated at the C-2 position. This can be used as a simple method for the identification of co-ordinated histidine residues in metalloproteins employing only minimal chemical modifications. 

The NMR-spectra of the human and the bovine enzymes are very similar. This is consistent with the significant homology in the amino acid sequences The similarity is such, that it was predicted by NMR-spectroscopists, that all six histidine residues are conserved. Furthermore, CujZuj-SOD from baker s yeast and from wheat germs also have conserved histidine residues, revealing considerable active-site structural homology between mammalian SOD s and the former two species. 

Cadmium-113 nuclear magnetic resonance studies of the cadmium substituted bovine superoxide dismutase were carried out Only a very small chemical-shift difference between the 2 Cd(Il) protein (Cd(II) is bound to the zinc site and the copper site is unoccupied) and the 2 Cd(ll)—2 Cu(I) enzyme (analogous to the reduced form of the native protein) was found. This was interpreted in that the imidazolate bridge is protonated at the Cu site after reduction. 

Electron-nuclear double resonance (ENDOR) is able to recover hyperfine and quadrupole information from incompletely resolved EPR-spectra. It was applied to native, azido- and cyano-derivatives of bovine Cu Zn superoxide dismutase . The ENDOR response of azido-SOD has led to the suggestion, that four nearly equivalent nitrogens surround the copper. The response of the native form was too complex for interpretation. The ENDOR response of the cyano form revealed that 

Extended X-ray-absorption fine structures (EXAFS) on copper and zinc K-edge were measured in aqueous solution After reduction of copper, it is co-ordinated to 

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