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 g_zz and A_zz 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.

Keywords:

• W-band ENDOR
• ESEEM
• metal ion coordination
• Cu²⁺ inhibition
• restriction endonuclease

Additional information

Funding

This work was supported by a National Science Foundation grant to Sunil Saxena and Linda Jen-Jacobson [grant number MCB 1157712]; preparation of purified EcoRI and EcoRI–DNA-Cu²⁺ complexes was supported by a National Institutes of Health MERIT grant to Linda Jen-Jacobson [grant number 5R37-GM029207]; the Bruker Elexsys E680 spectrometer was purchased with funds from a National Institute of Health grant [grant number 1S10RR028701].