Applications of electron paramagnetic resonance spectroscopy to study interactions of metalloenzymes with Cu(II) ions

In the past, the method of reconstitution was used to investigate the interaction between metalloenzymes (containing Zn(II)) and metal ions. In this paper, electron paramagnetic resonance (EPR) has been employed to firstly study the direct interactions between Bacillus subtilis neutral proteinase (BSNP), nuclease P1 and Cu(II) ions added in aqueous solution, respectively. These results show that a dynamic equilibrium exists between the Zn(II) in the active site of native enzymes and the added Cu(II), the added Cu(II) partly replaces the Zn(II), forming Cu(II)-enzyme derivatives. As a result, the activity of the native enzymes is influenced. The influences of pH value on this kind of interaction have also been investigated, and the results demonstrate that the change of pH value has little influence on the system of nuclease P1, but has remarkable influence on BSNP. We firstly obtained the EPR spectra for Cu(II)-enzyme derivatives. In addition, the derivative of Cu(II)-BSNP exists in the solution with two different conformations (I type g(parallel)=2.34, A(parallel) (mT)=13.4; II type g(parallel)=2.25, A(parallel) (mT)=16.1), and this two conformations exchanged each other depending on pH.