A Systematic Approach for Studying Cu Imidazole Interactions Using Multi-Frequency ESEEM: Application to Cu(II)-Bleomycin and Model Systems

Many copper containing proteins exhibit well defined ESEEM signals detected at X-band and C-band. In these systems the Cu(II) ion is coordinated to one or several histidine residues. The main sharp features measured in the ESEEM spectra originate from the interaction o f the unpaired electron with the remote nitrogen nucleus of the histidine ring. The relative intensities of these features contain information about the orientation of the NQI-tensor in the molecular axis frame as defined by the principal axes of the g-matrix. This information can be related to the orientation of the imidazole ring in the complex. We present a systematic approach to determine the constraints of the Euler angles a, flt / of the NQI-tensor in the g-matrix principal axis system. The First step is to analyze the intensity ratios of the quadrupole peaks and the line shape o f the double quantum feature measured on the canonical positions in the EPR spectrum. This will lead to a constraint in the angles (a , J3) as well as the effective hyperfine interaction. This information is further refined using spectra on other orientation selective positions. We have applied this method to Cu(ll)Bleomycin and two model compounds: Cu(II)-Pypep and Cu(H)-PMA of which we have determined the principal quadrupole values and the orientation of the quadrupole tensor with respect to the g matrix axis system.