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Cu Zn SOD O
2
+
2
+
+
•−
→
Cu Zn SOD O
+
2
+
+
k
≈ ×
2 10
9
/M/s
(4.22)
2
2
16
Cu Zn SOD O
+
2
+
+
•−
(
+
2
H
+
)
→
Cu Zn SOD H O
2
+
2
+
+
k
≈ ×
2 10
9
/M/s
.
2
2
2
17
(4.23)
Initial studies at very high concentrations of
O
•−
showed
K
m
should be
greater than 5 mM
O
•−
if the saturation mechanism was involved [31]. It was
also suggested the actual measured rate constants must be slower than
2 × 10
9
/M/s, considering the surface area differential between
O
•−
and cu-Zn-
SOD (∼1 : 150) [88, 93]. The measured “diffusion-controlled” rate constant may
be from the electrostatic guidance of the negatively charged
O
•−
into the posi-
tively charged active site [88, 90, 93]. This suggestion was supported by pulse
radiolysis experiments carried on various mutations of positively charged Arg
at the entrance of the active site as a function of ionic strength and pH (Fig.
4.4) [89]. The rates were decreased by neutralization and reversal of the posi-
tive charge on Arg (Fig. 4.4). The effect of charge was also supported by the
pH dependence behavior in which the protonation of anionic oxygen in
glutamate/aspartate recovered the decrease in rate due to the negative charge.
Furthermore, neutralization of the negative charge near the active site resulted
in faster enzyme activity than the wild-type enzyme [91]. A detailed examina-
tion of reaction (4.22) using a pulse radiolysis technique demonstrated that
enzymatic reduction and concomitant superoxide oxidation were pH indepen-
dent. However, reaction (4.23) was pH dependent to yield the overall pH
dependence of cu-Zn-SOD on the dismutation of superoxide [92].
4.1.4.1 Manganese Superoxide Dismutase.
MnSODs were first discovered
in 1970 [94]. Dimer and tetramer forms of MnSODs with a single manganese
atom per subunit have been determined. Bacteria and eukaryotes usually
contain dimer and tetramer forms, respectively [31]. In the structure of
MnSODs, manganese is bound in a trigonal bipyramidal geometry to four
ligands from the protein (three histidines and an aspartic acid residue) and a
fifth ligand from the solvent. It has been assumed that the solvent ligand is in
hydroxide and protonated forms under oxidized and reduced conditions,
respectively. The active sites and structures of three MnSODs (human,
Esch-
erichia coli
, and
Deinococcus radiodurans
) have been studied in detail [95-97].
Due to the high sequence structure homology of iron superoxide dismutase
(FeSOD) and MnSOD, iron instead of manganese in the active form can be
incorporated into MnSOD. For example, the replacement of iron in
E. coli
MnSOD
in vivo
under anaerobic conditions has the ability to inactivate the
enzyme [31, 98].
The mechanism of MnSOD dismutation is complex [99]. The kinetics results
displayed a “burst phase” and a “zero-order” phase rather than a first-order
phase at sufficiently high ratios of
[
. The first-order disappear-
ance of
O
•−
has been seen in other SODs. Therefore, MnSOD in its reduced
form has been suggested to react with two concomitant pathways [100-102].
O
•−
] : [
MnSOD
]
2
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