title: Metalation states versus enzyme activities of Cu, Zn-superoxide dismutase probed by electrospray ionization mass spectrometry.
abstract: 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 3Zn-, and Cu 2Zn 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 % (63)Cu and (68)Zn stable isotopes. It was found that (1) the Cu 4-dimer possessed 84% 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 3Zn-, and Cu 2Zn 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 2Zn 2-dimer comparable to the native enzyme.
authors.affiliation: Laboratory of Protein Profiling and Functional Proteomics, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan
authors.forename: Yuzo
authors.initials: Y
authors.lastname: Yamazaki
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Takao
mesh_terms: D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013482:Superoxide Dismutase / Q000032:analysis* / Q000378:metabolism*
chemical_list: D013482:Superoxide Dismutase