Electron paramagnetic resonance studies on the copper(II) substituted alkaline phosphatase from Escherichia coli

Abstract

The binding of Cu 2+ to Escherichia coli alkaline phosphatase has been studied by electron paramagnetic resonance (EPR). The obtained data are consistent with the existence of two specific metal binding sites and additional sites for more loosely bound copper. The most striking observation has been made about the metal binding when the signals from about 2 equivalents of copper bound to the enzyme were studied as a function of pH. Two different forms of EPR signals were detected; they were interconverted with a p K value of approximately 7.6. Superhyperfine structure could be detected at pH 6.5 as well as at pH 8.0. In the lower pH range, the EPR spectrum of the 65Cu 2+-alkaline phosphatase complex possesses superhyperfine splitting which is well resolved in g | region and five nitrogen hyperfine lines can be detected. In the higher pH range the EPR spectra shows superhyperfine splitting with seven nitrogen hyperfine lines. The addition of phosphates, phosphonates, arsenates and arsonates induces major changes on the Cu 2+-alkaline phosphatase EPR spectra in the pH range 6.0 to 7.5. Such spectral alterations are absent in the higher pH ranges. The binding of only one equivalent of phosphate, phosphonate, arsenate or arsonate is responsible for the complete spectral changes in the lower pH range. Thiophosphate was capable to induce spectral changes in the lower and in the higher pH ranges. A very significant feature in the EPR spectrum obtained after thiophosphate titration is that reduction of the copper takes place. A number of metal-complexing agents tested causes perturbation of the spectroscopic properties of the Cu 2+-alkaline phosphatase complex. However, these spectral changes are quite different from those induced by the above described substances.