DNA cleavage mediated by copper superoxide dismutase via two pathways

摘要

The known action of Cu, Zn superoxide dismutase (Cu2Zn2SOD) that converts O-2 to O-2 and H2O2 plays a crucial role in protecting cells from toxicity of oxidative stress. However, the overproduction Of Cu2Zn2SOD does not result in increased protection but rather creates a variety of unfavorable effects, suggesting that too much Cu2Zn2SOD may be injurious to the cells. The present study examined the DNA cleavage activity mediated by a Cu,,SOD that contains 1-4 copper ions, in order to obtain an insight into the aberrant copper-mediated oxidative chemistry in the enzyme. A high SOD activity was observed upon metallation of the apo-form Of Cu2Zn2SOD with Cu(II), indicating that nearly all of the Cu(II) in the Cu,,SOD is as active as the Cu(II) in the copper site of fully active Cu2Zn2SOD. Using a supercoiled DNA as substrate, significant DNA cleavage was observed with the Cu,SOD in the presence of hydrogen peroxide or mercaptoethanol, whereas DNA cleavage with free Cu(II) ions can occur only < 5% under the same conditions. Comparison with other proteins shows that the DNA cleavage activity is specific to some proteins including the Cu,SOD. The steady state study suggests that a cooperative action between the SOD protein and the Cu(II)may appear in the DNA cleavage activity, which is independent of the number of Cu(II) in the Cu,SOD. The kinetic study shows that a two-stage reaction was involved in DNA cleavage. The effects of various factors including EDTA, radical scavengers, bicarbonate anion, and carbon dioxide gas molecules on the Cu,SOD-mediated DNA cleavage activity were also investigated. It is proposed that DNA cleavage occurs via both hydroxyl radical oxidation and hydroxide ion hydrolysis pathways. This work implies that any form of the copper-containing SOD enzymes (including Cu2Zn2SOD and its mutants) might have the DNA cleavage activity. (c) 2006 Published by Elsevier Inc.