Affinity inactivation of bovine Cu, Zn superoxide dismutase by hydroperoxide anion, HO2−

Proc. Natl. Acad. Sci. U.S.A.

Chock²,

Stadtman³

1990

304

187

Cu,Zn superoxide dismutase (Cu,Zn-SOD; EC 1.15.1.1) is known to be inhibited slowly by H202. Using EPR and the spin traps 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) and N-tert-butyl-a-phenylnitrone (PBN), we have shown that Cu,Zn-SOD catalyzes the formation of "free" -OH radicals from H202 in pH 7.6 bicarbonate buffer. Supporting evidence includes the following: (i) H202 and active Cu,Zn-SOD are required to yield si nt signals from spin- radical formation and it also exerts no effect on the signals of DMPO-OH adducts when added together with the CuZn-SOD. The capacity of Cu,Zn-SOD to generate "free" 'OH radicals from H202 may in part explain the biological damage associated with elevated intracellular SOD activity.

Section: Resultssupporting

confidence: 87%

Copper, zinc superoxide dismutase catalyzes hydroxyl radical production from hydrogen peroxide.

Proc. Natl. Acad. Sci. U.S.A.

Chock²,

Stadtman³

1990

304

187

“…Here we smnmarize our findings obtained from the studies with purified FALS Cu,Zn-SOD mutants. In addition to the usual superoxide dismutation activity, Cu,Zn-SOD is known to exhibit anion binding capacity (Mata de Freitas and Valentine, 1984;Rigo et al, 1977), inactivation by its own reaction product H202 (Fuchs and Borders, 1983;Hodgson and Fridovich, 1975a;Symonyan and Nalbandyan, 1972), the purported peroxidase activity (Hodgson and Fridovich, 1975b), and capacity to enhance nitration of protein tyrosine residues by peroxinitrite (Beckman et al, 1992). We also found that Cu,Zn-SOD, but not Mn-SOD, has free radical-generating activity (Yim et al, 1990;.…”

mentioning

confidence: 65%

Enhanced free radical generation of FALS-associated Cu,Zn-SOD mutants

Chock

et al. 1999

neurotox res

Familial amyotrophic lateral sclerosis (FALS) is an inherited disorder of motor neurons, which is associated with missense mutations in the Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene. Mice from the G93A transgenic line were reported to develop a syndrome of FALS. The fact that the symptoms occurred against a background of normal mouse Cu,Zn-SOD activity suggests that dominant, gain-of-function mutations in SOD play a role in the pathogenesis of FALS. We investigated the nature of this gain-of-function of FALS mutants. We have previously reported that Cu,Zn-SOD has the free radical-generating function in addition to normal dismutation activity. These two enzymic activities were compared by using mutants (G93A and A4V) and the wild-type Cu,Zn-SOD prepared by recombinant method. Our results showed that the wild-type, G93A, and A4V enzymes have identical dismutation activity. However, the free radical-generating function of the G93A and A4V mutants, as measured by the spin trapping and EPR method, is enhanced relative to that of the wild-type enzyme (wild type < G93A < A4V), particularly at lower H(2)O(2) concentrations. This is due to the decrease in the K(m) value for H(2)O(2), wild-type > G93A > A4V. The catalytic activity to generate free radicals is correlated to the clinical severity of the disorder induced by these mutant enzymes. Furthermore, we found that intact FALS mutants failed to enhance tyrosine nitration. Together our results indicate that the amyotrophic lateral sclerosis symptoms are not caused by the reduction of Cu,Zn-SOD dismutation activity with the mutant enzymes; rather, it is induced in part by enhancement of the free radical-generating function.

“…In addition to the usual superoxide dismutation activity, Cu,Zn-SOD is known to exhibit anion binding capacity (9,10), inactivation by its own reaction product H202 (11)(12)(13), and the purported peroxidase activity (14). Here we summarize our findings that Cu,Zn-SOD has free radical ('OH and scavenger-derived radicals)-generating activity (15,16).…”

mentioning

confidence: 78%

Pro-oxidant activity of Cu,Zn-superoxide dismutase

Chock

et al. 1998

AGE

Whereas Cu,Zn-superoxide dismutase (Cu,Zn-SOD) is an essential antioxidant enzyme for superoxide dismutation in vivo(1 ), many reports have shown that an elevated level of Cu,Zn-SOD induces cell killing (2, 3), increases lipid peroxidation (4,5), and interferes with the transport of neurotransmitters (4, 6). All of these observations are archetypical results inducible by oxygen radicals. Cu,Zn-SOD (7) and FALS mutants (8) are known to develop neurodegeneration. These findings suggest that under certain conditions Cu,Zn-SOD may behave directly or indirectly as a pro-oxidant.In addition to the usual superoxide dismutation activity, Cu,Zn-SOD is known to exhibit anion binding capacity (9, 10), inactivation by its own reaction product H202 (11-13), and the purported peroxidase activity (14). Here we summarize our findings that Cu,Zn-SOD has free radical ('OH and scavenger-derived radicals)-generating activity (15,16). TheactiveCu,Zn-SOD can catalyze the generation of free 9 radicals from H202. In the presence of H202 and radical scavengers, especially with anionic character, both free 9 radicals and scavenger-derived radicals are produced (15, 16). We also found that the free radical-generating function of several FALS-associated Cu,Zn-SOD mutants is enhanced relative to that of the wild-type enzyme, while the dismutation activities remain unchanged (17,18).To monitor the production of free radicals, two spin traps, 5,5-dimethyl-l-pyrroline N-oxide (DMPO) and Ntert-butyl-c~-phenylnitrone (PBN), were used in our studies (15) to convert transient free radicals to stable free radicals adducts according to the following reactions:The nature of the trapped free radical was identified by EPR spectroscopy. When O2-" (dissolved KO 2 in dried dimethylsulfoxide) was added to the NaHCOJCO 2 buffer at pH 7.6 containing 100 mM DMPO, EPR OH adducts. DMPO-OH formation also required active Cu,Zn-SOD, because heat-inactivated Cu,Zn-SOD or Mn-SOD failed to produce these radical adducts. The direct addition of H202 in place of O2-" produced similar results. In the presence of .OH radical scavengers with anionic character such as HCO 2 or N3-, DMPO adducts of the scavenger-derived radicals, DMPO-CO2-" or DMPO-N3", were observed in addition to DMPO-OH. The concentration ratio between DMPO-OH and DMPO-scavenger-derived radical adducts observed in these experiments reflects competition reactions between DMPO and scavengers for free "OH radicals. Surprisingly, with neutral'OH radical scavengers, such as ethanol, the formation of DMPO-hydroxyethyl radical adducts was insignificant and the concentration of DMPO-OH formed was unaffected. In contrast, with a different spin trap, PBN, in place of DMPO, ethanol yields PBNhydroxyethyl radical adducts as expected in the competition reaction for free "OH radicals. These seemingly contradicting results may be caused by the different affinities of these spin traps for the positively charged active channel leading to the active site of Cu,Zn-SOD, where the'OH radicals are generated. To prove this hypo...