Cu,Zn Superoxide Dismutase: A Case of Metalloenzyme Catalysis in Which the Protein Moiety Plays a Major Role

Abstract

The structure and catalytic mechanism of Cu,Zn superoxide dismutase (SOD) are known from studies of the bovine enzyme (for review see refs. 1, 2). This enzyme consists of two identical subunits of 16 kD, each containing a cataly­tically active copper ion and a zinc ion, which shares a common ligand—the imidazole of His 61—with the copper. It has been demonstrated (3, 4) that the enzyme-catalyzed superoxide ion dismutation proceeds by a cyclic oxidation-reduction mechanism involving the copper ion: $$ ECu\left({II}\right)+O_2^-\xrightarrow{{{k_1}}}ECu\left(I\right)+{O_2} $$ (1) $$ ECu\left(I\right)+O_2^- +2{H^+}\xrightarrow{{{k_2}}}ECu\left({II}\right)+{H_2}{O_2} $$ (2) Our previous pulse-radiolysis work (4) has demonstrated that the reactions between O2 - and either reduced or oxidized forms of the enzyme are diffusion-limited and occur at the same rate of ~ 2 × 109 M -1 s-1 for each Cu site. No rate-limiting first-order process has been observed at the superoxide concen­tration accessible to pulse radiolysis. A Km ≃ 0.4 mM could be measured by a polarographic measurement (5). However, all detailed kinetic studies are performed at [O2 −] « Km because of technical limitations. Under these con­ditions the rate-limiting process is the binding of substrate to the enzyme active site. Previous results showed that the binding rates are independent of pH in the range between 5.0 and 9.5 (6, 7) and are unaffected by D2O (8), indicating that proton transfer is not a part of the binding process. This finding is in line with the identical, diffusion-controlled values of the rate constants measured for the individual redox reactions 1 and 2 (Eqs. 1 and 2). Under saturating conditions, which cannot be explored by current kinetic studies, proton transfer in Eq. 2 could become rate-limiting.