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

Abstract

Bovine erythrocyte superoxide dismutase (Cu(2)Zn(2)SODase; superoxide:superoxide oxidoreductase, EC 1.15.1.1) consists of two identical subunits each containing Cu(2+) and Zn(2+) in close proximity. We describe here electron spin resonance (ESR) and visible absorption spectroscopic studies of the zinc-free derivative of this protein, Cu(2)E(2)SODase (E = empty) over the pH range 6-10. The ESR spectrum of the zinc-free protein at 77 K is markedly pH dependent. At pH < 8.0 the ESR spectrum is axial in appearance. At pH > 8.0, the lineshape becomes increasingly distorted with increasing pH until, at pH = 9.5, the spectrum is very broad and resembles that of the four-copper derivative Cu(2)Cu(2)SODase and of model imidazolate-bridged binuclear Cu(II) complexes. ESR spectra at 30 degrees C are also consistent with formation of Cu(II)-Im-Cu(II). A plot of changes in the signal amplitude of g perpendicular for Cu(2)E(2)SODase as a function of pH gives an apparent pK(a) of 8.2 for the transition. The long-wavelength absorption with lambda(max) = 700 nm characteristic of Cu(2)E(2)SODase shifts with increasing pH to 800 nm and the resulting visible spectrum is identical to that of Cu(2)Cu(2)SODase. All of the above-mentioned spectroscopic changes induced by additions of NaOH are reversed when the pH is decreased with HNO(3), although the approach to equilibrium is slow in the latter case. The results of these experiments are consistent with a reversible, pH-dependent migration of Cu(2+) from the native copper site of one subunit of the zinc-free protein to the empty zinc site of another subunit. By contrast, native protein, Cu(2)Zn(2)SODase, and the four-copper protein, Cu(2)Cu(2)SODase, show no variation in visible or ESR spectral properties in this pH range. Some previous results concerning the activity of Cu(2)E(2)SODase and its thermal stability are reexamined in light of these new findings.