Structure

The x-ray crystal structure of the oxidized form of CuZnSOD from bovine erythrocytes shows a protein consisting of two identical subunits held together almost entirely by hydrophobic interactions.loo-I02 Each subunit consists of a flattened cylindrical barrel of {3-pleated sheet from which three external loops of irregular structure extend (Figure 5.15). The metal-binding region of the protein binds Cu II and Zn II in close proximity to each other, bridged by the imidazolate ring of a histidyI side chain. Figure 5.16 represents the metal-binding region. The CuII ion is coordinated to four histidyl imidazoles and a water in a highly distorted square-pyramidal geometry with water at the apical position. The ZnII ion is coordinated to three histidyl imidazoles (including the one shared with

copper) and an aspartyl carboxylate group, forming a distorted tetrahedral geometry around the metal ion.
 

One of the most unusual aspects of the structure of this enzyme is the occurrence of the bridging imidazolate ligand, which holds the copper and zinc ions 6 Aapart. Such a configuration is not unusual for imidazole complexes of metal ions, which sometimes form long polymeric imidazolate-bridged structures.

However, no other imidazolate-bridged bi- or polymetallic metalloprotein has yet been identified.
 

The role of the zinc ion in CuZnSOD appears to be primarily structural. There is no evidence that water, anions, or other potential ligands can bind to the zinc, so it is highly unlikely that superoxide could interact with that site. Moreover, removal of zinc under conditions where the copper ion remains bound to the copper site does not significantly diminish the SOD activity of the enzyme. J10 However, such removal does result in a diminished thermal stability, i.e., the zinc-depleted protein denatures at a lower temperature than the native protein, supporting the hypothesis that the role of the zinc is primarily structural in nature. JJJ
 

The copper site is clearly the site of primary interaction of superoxide with the protein. The x-ray structure shows that the copper ion lies at the bottom of a narrow channel that is large enough to admit only water, small anions, and similarly small ligands (Figure 5.17). In the lining of the channel is the positively charged side chain of an arginine residue,S Aaway from the copper ion

and situated in such a position that it could interact with superoxide and other anions when they bind to copper. Near the mouth of the channel, at the surface of the protein, are two positively charged lysine residues, which are believed to play a role in attracting anions and guiding them into the channel. lIZ Chemical modification of these lysine or arginine residues substantially diminishes the SOD activity, supporting their role in the mechanism of reaction with superoxide. 100-10Z
 

The x-ray structural results described above apply only to the oxidized form of the protein, i.e., the form containing CUll. The reduced form of the enzyme containing CuI is also stable and fully active as an SOD. If, as is likely, the mechanism of CuZnSOD-catalyzed superoxide disproportionation is Mechanism I (Reactions 5.96-5.97), the structure of the reduced form is of critical importance in understanding the enzymatic mechanism. Unfortunately, that structure is not yet available.