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

<div><p>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 Cu<sup>2+</sup> binds outside the catalytic sites to a pair of new sites at H114 in each sub-unit, and inhibits Mg<sup>2+</sup>-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–(Cu<sup>2+</sup>)<sub>2</sub> complex. Cu<sup>2+</sup> binding results in two distinct components with different <i>g</i><sub>zz</sub> and <i>A</i><sub>zz</sub> values. X-band electron spin echo envelope modulation results indicate that both components arise from a Cu<sup>2+</sup> 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 Cu<sup>2+</sup> ions.</p></div>