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+)2complex. Cu2+binding results in two distinct components with differentgzzandAzzvalues. 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.
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA.
Ming
M
Ji
Division of Engineering and Applied Science, California Institute of Technology, Pasadena, CA, USA.
Likun
L
Tan
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA.
Linda
L
Jen-Jacobson
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA.
Sunil
S
Saxena
Cu2+ inhibition; ESEEM; W-band ENDOR; metal ion coordination; restriction endonuclease