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

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.