The pH dependence of apparent binding constants between apo-superoxide dismutase and cupric ions

Abstract

Apo-superoxide dismutase can bind four copper ions to two native copper- and two native zinc-binding sites. Four, stepwise, apparent binding constants of copper ions to the apo-enzyme were obtained from the equilibrium dialysis data at various pH values. These binding constants are pH dependent. In the low pH region, the binding of copper ions to the native copper binding sites has much larger apparent binding constants than to the native zinc sites. But the difference between apparent binding constants of copper ions to the native copper sites and those to the native zinc sites decreased with an increase of pH. At pH 10.0, it is impossible to distinguish between the binding of copper ions to the native copper sites and to the native zinc sites. The relationship between logarithm of apparent binding constants of copper sites and pH was linear with a slope of 2 in the pH region from 4.0 to 7.0. Above pH 7.0, apparent binding constants at the copper sites were almost pH independent. These results indicate that copper ions compete with two protons in the native copper-binding sites and that p K a values of the protons were almost 7. From apparent binding constants of copper ions to the apo-enzyme at pH 7.0 and 10.0, theoretical EPR-detectable copper contents were calculated and compared with EPR data obtained by J. S. Valentine, M. W. Pantoliano, P. J. McDonnell, A. R. Burger, and S. J. Lippard ( Proc. Nat. Acad. Sci. USA, 1979 , 76, 4245–4249).