Vanadyl, VO(2+), was used as an electron paramagnetic resonance (EPR) spin label to study the metal-binding properties of bovine copper, zinc-superoxide dismutase (Cu,Zn-SOD). The vanadyl SOD derivative was prepared in 0.10 M HEPES, pH 7.4, at a ratio of 2:4:1 of VO(2+):dithionite:apo-SOD, and under a nitrogen atmosphere. X-band EPR titrations indicated that two VO2+ ions bound strongly to the protein dimer establishing a 1:1 stoichiometry per subunit. A comparison of the EPR parameters g-parallel and A-parallel obtained for the protein derivative with those previously reported for model compounds indicated that VO(2+) is bound at the native copper site. Exposure of the vanadyl SOD derivative to air for 90 min showed a decrease of approximately 35% in the peak intensity of the EPR spectrum indicating slow oxidation of VO(2+) in the protein derivative. X-band EPR titrations of the zinc-only derivative (E,Zn-SOD, E = empty) with VO(2+) showed that the occupation of the native zinc site decreased the binding of VO(2+) to the native copper site. Addition of VO(2+) to native protein under similar experimental conditions gave a characteristic Cu(2+) EPR spectrum ruling out adventitious binding of VO(2+) to amino acid residues not at the active site of bovine Cu,Zn-SOD. The peak potential values measured by cyclic voltammetry at pH 4.0 for VO,E-SOD and native Cu,Zn-SOD were +425 mV and +300 mV, respectively. The SOD activity of VO,E-SOD was examined by using the indirect xanthine oxidase/cytochrome c assay method. However, the derivative was found to be SOD inactive. We conclude that vanadyl can be used to probe the metal binding properties of the copper site in Cu,Zn-SOD. ?
Showing the most recent 10 out of 368 publications
