SOD,CuZn

The CuZn-SOD activity is completely inhibited by cyanide, and also partly by azide. The Cu2+ and Zn2+ ions can be substituted by other metal ions such as Co2+, Ni2+ etc., but the activity is found only when those substituted enzymes retain Cu2+ in the native site (Table 10.1). 

Subunit structure and homodimer homodimer / homotetramer homodimer/homotetramer 

Amino acid sequence No homology with Mn-/Fe-SOD High homology with Fe-SOD High homology with Mn-SOD 

Specific activity (McCord-Fridovich unit/mg enzyme) 8,000 6,000 6,000 

Rate constant with Of (M 1 s 1) 2X109 2X109 2X109 

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Figure 5.5 CuZn-SOD active sites in (A) reduced and (B) oxidized forms as described in reference 30.

Ogawa et al. (1997) investigated the formation of H202 and the superoxide radical (02 ) in spinach (Spinacia oleracea) hypocotyls with the use of histochemical stains. Nitroblue tetrazolium (NBT) is used to detect 02 radicals. The colored reaction product formazan was only detected in the vascular tissue of developing spinach hypocotyls if CuZn-superoxide dismutase (CuZn-SOD E.C. 1.15.1.1) was inhibited by DDC... 

The one isolated from bovine blood contains Cu in its reaction center as well as Zn which appears not to take part in the dismutation process. The other two types of SOD contain either Fe or Mn. The CuZn SOD has been found only in eukariotic cells, the Fe SOD only in prokaryotic cells, and the Mn SOD in both (Fee 1981). Aqua-Mn2+ cannot be reduced by 02, but is forms a complex that dismutates giving rise to H202 and 02 (Jacobsen et al. 1997). Such intermediates may also play a role in Mn SOD. 

Nevertheless, with respect to the catalysis of the dismutation of O2, little difference has been found among the three types of SOD.I0) The reaction rate constants between O2 and the enzyme are about 2X109 M- s at neutral pH. However, although the catalytic rate of the CuZn-SOD is constant between pH 5 and 9.5, those of the Fe- and Mn-SODs become progressively lower as the pH is raised above 8.5. 

Chloroplast type of CuZn-SOD isozyme is localized in the chloroplast stroma of most plants, whereas Fe-SOD occurs in chloroplasts of Euglena, the moss Marchantia polymorpha and several species of seed plant. So far Mn-SOD has not been detected in chloroplasts in a soluble form but occurs in a thylakoid membrane-bound form.23,24) The cytosolic CuZn-SOD, which is distinguishable from the chloroplastic CuZn-SOD in terms of amino acid sequence, occurs in cell compartments other than chloroplasts and in nonphotosynthetic tissues.14)... 

Amino acid and nucleotide sequences of CuZn-SOD have been determined for enzymes from many organisms. The amino acid residues of the metal ligands and the cysteine residues which form disulfide bridge are conserved in all CuZn-SODs so far sequenced. Furthermore, the charged arginine and lysine residues which participate in the catalytic function for attracting anionic O2 to the Cu site are also conserved (Fig. 10.3). The amino... 

Fig. 10.3 Amino acid sequences of CuZn-SODs from various organisms.31 Yeast, Saccharomyces cerevisiae Neurospora, Neurospora crassa Arabido., Arabidopsis thaliana Cyt, cytosolic CuZn-SOD Chi, chloroplastic CuZn-SOD. Amino acid residue number is based on that of human CuZn-SOD. Residues conserved in all species are boxed.

The three-dimensional structures have been determined for bovine (2.0 A resolution), 41,42) yeast (2.5 A),43 spinach (2.0 A)44 and human (2.5 A)45 CuZn-SODs. The structure of cobalt-substituted bovine CuCo-SOD (2.0 A) has also been determined.46 The overall structure of the enzyme is a barrel structure consisting of eight antiparallel fi strands (46% of total amino acid residues) and three loops (48%). The two large loops are fixed by an internal disulfide bond and contain ligating amino acid residues to the Cu and the Zn. The content of a helix is only 5%, and it is localized in the loop region (Fig. 10.4). 

Fig. 10.4 Chain fold of bovine CuZn-SOD.4l) The p strands are shown by arrows and the disulfide bridge by a zig-zag. The Cu and Zn lie at the bottom of the active-site cavity.

Fig. 10.5 Active site of spinach CuZn-SOD.44 The geometry of the Zn ligands is approximately tetrahedral. The Cu ligands form a distorted square plane.

Amino acid residue number of CuZn-SOD is based on that of human CuZn-SOD consisting of 153 residues throughout. The residue numbers of Fe— and Mn—SODs are those of each enzyme. 

Reproduced from J. Biochem., 91, 895 (1982)) CuZn-SOD was added, respectively. 

The catalytic mechanism of CuZn-SOD involves alternate reduction and reoxidation of the Cu ion during successive interactions with 0265) ... 

The dismutation rate of 05 catalyzed by CuZn-SOD, 2X109 M-1 s l, is 10% of the rate of diffusion-limited reaction expected for a small molecule (05) and a macromolecule (SOD), whereas the Cu site comprises only 0.1% of the total surface area of CuZn-SOD. This indicates that the interaction of 05 with the Cu site is facilitated 100-fold, for which an electrostatic interaction is responsible. 

Fig. 10.10 Surroundings of the active site of bovine CuZn-SOD (A)42) and schematic drawing of its active cavity (B)90). In (A), main chain shown in black bonds, ligand side chain open bonds, and other side chains with solid atoms and open bonds.

When the CH concentration is below saturation, the rate-limiting step in the catalytic process of CuZn-SOD is the diffusion of O2 to the active site channel and its binding to the Cu2+. After entering the active cavity, O2 diffuses to the coordination sphere of the Cu. This step involves a displacement of the Cu2+-bound water molecule by O2 and the formation of bonds among O2, the Cu2+ ion and Arg-143. The reduction of the Cu2+ is... 

The reaction mechanisms of Fe-SOD and Mn-SOD are not yet as well characterized as those of CuZn-SOD, but the ligands of the metals have been confirmed to be three histidyl immidazoles and one aspartyl carboxylate. The Fe- and Mn-SODs undergo alternate reduction and reoxidation during the catalytic cycle, similar to CuZn-SOD, at a rate constant of 2X109 M s l. The electron transfer to O2 from the metals proceeds through an outer sphere mechanism for Fe- and Mn-SODs, while it proceeds via an inner sphere mechanism for CuZn-SOD. Fe- and Mn-SODs exhibit substrate saturation77 as does CuZn-SOD. 

Mn-SODs causes decrease in the activity, indicating that lysine residues are implicated in the electrostatic field around the active site.81 Thus, Mn-SOD and Fe-SOD exhibit a guiding electrostatic field around its active site in a way similar to that of CuZn—SOD.68 ... 

CuZn-SOD is inactivated by H2O2 through the following mechanism H2O2 first reduces the enzyme-Cu2+ to the Cu+ form (reaction 10.15), then the enzyme-Cu+ reacts with H2O2 to form a strong oxidant at the active site by a Fenton-like reaction (reaction 10.16). The oxidant destroys the Cu-ligated His-63.84 ... 

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