Electron spin echo modulation study of the Type I copper protein rusticyanin and its mutant variant His85Ala - Implications for the general analysis of weak N-14 superhyperfine interactions

Electron spin echo modulation (ESEEM) spectra were recorded at 500 MHz intervals for the Type I protein rusticyanin and its engineered variant His85Ala, which lacks one of the two imidazole ligands to copper. The Zeeman and g-value dependence of the spectral peaks were systematically mapped and accurately predicted that low intensity lines in the wild type (w.t.) spectrum were combination lines, as verified in the His85Ala spectra. Analysis reveals that the w.t. ESEEM spectra are attributable to a pair of HisN(epsilon) only, and the NQI parameters e(2)Qq and eta are 1.37 +/- 0.03 MHz and 0.90 +/- 0.05 for both HisN(epsilon) in the w.t. protein. The nuclear hyperfine interaction is estimated as 1.8 MHz, and includes a dipolar interaction of 0.5 MHz. The principal difference between the two imidazole nitrogen couplings is manifest in the dispersion of the combination lines, which suggests non-equivalent orientations of the respective superhyperfine tensors (or tilt of the imidazole ring). The protocol demonstrates a general procedure for accurately determining superhyperfine parameters from powder ESEEM spectra. Finally, peak shifts among ESEEM spectra of His85Ala-X (X = H2O, Cl-, Br-) demonstrate that the imidazole ligand powder superhyperfine spectrum is sensitive to chemical effects in the metal's ligand sphere, thus suggesting that the method may be used for evaluation in molecular design.