Insights into copper coordination in the EcoRI–DNA complex by ESR spectroscopy

Abstract

The EcoRI restriction endonuclease requires one divalent metal ion in each of two symmetrical and identical catalytic sites to catalyse double-strand DNA cleavage. Recently, we showed that Cu2+ binds outside the catalytic sites to a pair of new sites at H114 in each sub-unit, and inhibits Mg2+ -catalysed DNA cleavage. In order to provide more detailed structural information on this new metal ion binding site, we performed W-band (~94 GHz) and X-band (~9.5 GHz) electron spin resonance spectroscopic measurements on the EcoRI–DNA–(Cu2+ )2 complex. Cu2+ binding results in two distinct components with different gzz and Azz values. X-band electron spin echo envelope modulation results indicate that both components arise from a Cu2+ coordinated to histidine. This observation is further confirmed by the hyperfine sub-level correlation results. W-band electron nuclear double resonance spectra provide evidence for equatorial coordination of water molecules to the Cu2+ ions.

Probing Axial Water Bound to Copper in Tutton Salt Using Single Crystal 17O-ESEEM Spectroscopy

“…88 Likewise, a recent W-band ENDOR investigation on the ECoR1 restriction endonuclease DNA complex detected 1 H couplings to copper which were tentatively assigned as arising from both equatorial and axial bound waters. 89 The present report demonstrates the continuing capability of X-band ESEEM spectroscopy to the direct study of weak 17 O couplings from remote copper ligands, which may be useful in confirming these findings.…”

“…Previous 2-pulse ESEEM techniques have been able to analyze small proton and deuteron hyperfine interactions in metalloproteins, resulting in good but only indirect evidence for the existence of coordinated axial water . Likewise, a recent W-band ENDOR investigation on the ECoR1 restriction endonuclease DNA complex detected 1 H couplings to copper which were tentatively assigned as arising from both equatorial and axial bound waters . The present report demonstrates the continuing capability of X-band ESEEM spectroscopy to the direct study of weak 17 O couplings from remote copper ligands, which may be useful in confirming these findings.…”

The Advantages of EPR Spectroscopy in Exploring Diamagnetic Metal Ion Binding and Transfer Mechanisms in Biological Systems

“…In other studies, which were not discussed here, diamagnetic metal sites have been resolved by EPR spectroscopy by replacing the diamagnetic metal ions with a similar paramagnetic metal ion. For example, by replacing Mg(II) or Zn(II) with Mn(II) or Cu(II) [105,106], which possess similar properties, and, in most cases, they bind to the biomolecule similarly to the diamagnetic metal ion.…”

An Undergraduate Experiment To Explore Cu(II) Coordination Environment in Multihistidine Compounds through Electron Spin Resonance Spectroscopy

“…ESR can also be used to monitor radical formation in chemical reactions and in degradation processes. As a biophysical tool, ESR has been extensively used for probing structure and dynamics of proteins and nucleic acids ,,− as well as protein–nucleic acid complexes. − Hence, ESR plays an important role in advancing the field of biophysics. The wide-reaching applications and unique information about electron density and chemical structure provided by ESR make it an important magnetic resonance spectroscopy technique to be covered in the undergraduate chemistry curriculum.…”