Metalation States versus Enzyme Activities of Cu, Zn-Superoxide Dismutase Probed by Electrospray Ionization Mass Spectrometry

摘要

Cu, Zn-superoxide dismutase (SOD-1), an enzyme that catalyzes the disproportionation reaction of superoxide to produce oxygen and hydrogen peroxide, thereby protecting cells from oxidative stress, is a homodimer that coordinates one copper and one zinc ion per monomer. Cu~(2+) and Zn~(2+) ions play important roles in enzyme activity and structural stability, respectively. In addition, dimer formation is also essential for fulfilling the function of SOD-1. We here report on the reconstitution and enzyme activities of several metalation states of SOD-1 (Cu_(4)-, Cu_(3)Zn-, and Cu_(2)Zn_(2)-homodimers). Each metalation state of the reconstituted SOD-1 could be unambiguously differentiated by electrospray ionization mass spectrometry, the metal ions of which had been completely replaced by 99 atom percent ~(63)Cu and ~(68)Zn stable isotopes. It was found that (1) the Cu_(4)-dimer possessed 84percent of the activity of the native enzyme, (2) the Cu-site resisted being coordinated with Zn~(2+) ions while the Zn-site could be bound with Cu~(2+) ions, and (3) the simultaneous addition of the Cu~(2+) and Zn~(2+) ions to generate a fully metalated form produced the multiply metalated SOD-1 (Cu_(4)-, Cu_(3)Zn-, and Cu_(2)Zn_(2)-dimers), which were clearly distinguishable from one another by the use of the stable isotopes, while the sequential addition of Zn~(2+) followed by the Cu~(2+) ion predominantly produced a Cu_(2)Zn_(2)-dimer comparable to the native enzyme.