Insights on Copper Coordination and Reactivity of Endonuclease EcoRI by ESR Spectroscopy and Modeling

Abstract

Restriction endonuclease EcoRI cleaves a specific six base pair sequence of DNA in the presence of some divalent metal ions such as Mg2+. Cu2+, on the other hand, does not catalyze the cleavage of DNA by EcoRI. To understand the functional difference between Cu2+ and Mg2+, electron spin resonance (ESR) spectroscopy was used to determine the local environment of Cu2+ in EocRI. Two Cu2+ components are observed from the continuous wave-ESR spectrum. Electron spin echo envelope modulation (ESEEM) spectra under different external magnetic fields have contributions from both of the two Cu2+ components (except at a low magnetic field). Both components contain peaks below 2 MHz and broad peaks at ∼4 MHz. A peak at ∼14 MHz corresponding to proton ESEEM is also observed in each spectrum. To quantitatively analyze the spectra, we developed a theoretical simulation package to simulate these spectra. Based on the simulation, both of the two Cu2+ components have a 14N of histidine imidazole coordinated to Cu2+. The peaks below 2 MHz and at ∼ 4 MHz are assigned to the nuclear quadrupole interaction and the double quantum transitions of the remote 14N of histidine imidazole, respectively. We discovered H114 of EcoRI coordinates to the Cu2+, based on distance measurements using double electron electron resonance spectroscopy. And molecular dynamics simulation confirms the coordination of H114 to Cu2+. These results help us to get an understanding of the different coordination environments between Cu2+ and Mg2+ in EcoRI-DNA, and thus to explain the functional difference between them. This work supported by NSF.