Enzymatic Activity and Mechanism

The mechanism of superoxide disproportionation catalyzed by CuZnSOD is generally believed to go by Mechanism I (Reactions 5.96-5.97), i.e., reduction of CuII to CuI by superoxide with the release of dioxygen, followed by reoxidation of CUI to CUll by a second superoxide with the release of HOz- or HzOz. The protonation of peroxide dianion, Oz z-, prior to its release from the enzyme is required, because peroxide dianion is highly basic and thus too unstable to be released in its unprotonated form. The source of the proton that protonates peroxide in the enzymatic mechanism is the subject of some interest.
 

Reduction of the oxidized protein has been shown to be accompanied by the uptake of one proton per subunit. That proton is believed to protonate the bridging imidazolate in association with the breaking of the bridge upon reduction of the copper. Derivatives with CoII substituted for ZnII at the native zinc site have been used to follow the process of reduction of the oxidized CuII form to the reduced Cu1form. The COIl in the zinc site does not change oxidation state, but acts instead as a spectroscopic probe of changes occurring at the native zincbinding site. Upon reduction (Reaction 5.102), the visible absorption band due to CoII shifts in a manner consistent with a change occurring in the ligand environment of CoII. The resulting spectrum of the derivative containing CuI in the copper site and COIl in the zinc site is very similar to the spectrum of the derivative in which the copper site is empty and the zinc site contains COlI. This result suggests strongly that the imidazolate bridge is cleaved and protonated and that the resulting imidazole ligand is retained in the coordination sphere of COIl (Reaction 5.102).

The same proton is thus an attractive possibility for protonation of peroxide as it is formed in the enzymatic mechanism (Reactions 5; 103 and 5. 104).

Attractive as this picture appears, there are several uncertainties about it. For example, the turnover of the enzyme may be too fast for protonation and deprotonation of the bridging histidine to occur. 113 Moreover, the mechanism proposed would require the presence of a metal ion at the zinc site to hold the imidazole in place and to regulate the pKa of the proton being transferred. The observation that removal of zinc gives a derivative with almost full SOD activity is thus surprising and may also cast some doubt on this mechanism. Other criticisms of this mechanism have been recently summarized. 

Studies of CuZnSOD derivatives prepared by site-directed mutagenesis are also providing interesting results concerning the SOD mechanism. For example, it has been shown that mutagenized derivatives of human CuZnSOD with major differences in copper-site geometry relative to the wild-type enzyme may nonetheless remain fully active. 114 Studies of these and similar derivatives should provide considerable insight into the mechanism of reaction of CuZnSOD with superoxide.