pH-dependent migration of copper(II) to the vacant zinc-binding site of zinc-free bovine erythrocyte superoxide dismutase

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Abstract
Bovine erythrocyte superoxide dismutase (Cu2Zn2SODase; superoxide:superoxide oxidoreductase, EC 1.15.1.1) consists of 2 identical subunits each containing Cu2+ and Zn2+ in close proximity. ESR and visible absorption spectroscopic studies of the Zn-free derivative of this protein, Cu2E2SODase (E = empty) over the pH range 6-10 were described. The ESR spectrum of the Zn-free protein at 77.degree. K was markedly pH dependent. At pH < 8.0 the ESR spectrum was axial in appearance. AtpH > 8.0, the lineshape becomes increasingly distorted with increasing pH until, at pH = 9.5, the spectrum was very broad and resembled that of the 4-Cu derivative Cu2Cu2SODase and of model imidazolate-bridged binuclear Cu(II) complexes. ESR spectra at 30.degree. C were also consistent with formation of Cu(II)-Im-Cu(II). A plot of changes in the signal amplitude of g.perp. for Cu2E2SODase as a function of pH gave an apparent pKa of 8.2 for the transition. The long-wavelength absorption with .lambda.max = 700 nm characteristic of Cu2E2SODase shifts with increasing pH to 800 nm and the resulting visible spectrum was identical to that of Cu2-Cu2SODase. All of the above-mentioned spectroscopic changes induced by additions of NaOH were reversed when the pH was decreased with HNO3, although the approach to equilibrium was slow in the latter case. The results of these experiments were consistent with a reversible, pH-dependent migration of Cu2+ from the native Cu site of 1 subunit of the Zn-free protein to the empty Zn site of another subunit. By contrast, native protein, Cu2n2SODase, and the 4-Cu protein, Cu2-Cu2SODase, show no variation in visible or ESR spectral properties in this pH range. Some previous results concerning the activity of Cu2E2SODase and its thermal stability were reexamined in light of these new findings.