A SYSTEMATIC-APPROACH FOR STUDYING CU IMIDAZOLE INTERACTIONS USING MULTIFREQUENCY ESEEM - APPLICATION TO CU(II)-BLEOMYCIN AND MODEL SYSTEMS

Many copper containing proteins exhibit well defined ESEEM signals det ected at X-band and C-band. Ln these systems the Cu(II) ion is coordin ated to one or several histidine residues. The main sharp features mea sured in the ESEEM spectra originate from the interaction of the unpai red electron with the remote nitrogen nucleus of the histidine ring. T he relative intensities of these features contain information about th e orientation of the NQI-tensor in the molecular axis frame as defined by the principal axes of the g-matrix. This information can be relate d to the orientation of the imidazole ring in the complex. We present a systematic approach to determine the constraints of the Euler angles alpha, beta, gamma of the NQI-tensor in the g-matrix principal axis s ystem. The first step is to analyze the intensity ratios of the quadru pole peaks and the line shape of the double quantum feature measured o n the canonical positions in the EPR spectrum. This will lead to a con straint in the angles (alpha, beta) as well as the effective hyperfine interaction. This information is further refined using spectra on oth er orientation selective positions. We have applied this method to Cu( II)-Bleomycin and two model compounds: Cu(II)-Pypep and Cu(II)-PMA of which we have determined the principal quadrupole values and the orien tation of the quadrupole tensor with respect to the g-matrix axis syst em.