Cysteine ligand vibrations are responsible for the complex resonance Raman spectrum of azurin

In the redox center of azurin, the Cu(II) is strongly coordinated to one thiolate S from Cys 112 and two imidazole Ns from His 46 and 117. This site yields a complex resonance Raman (RR) spectrum with >20 vibrational modes between 200 and 1500 cm(-1), We have investigated the effects of ligand-selective isotope replacements on the RR spectrum of Pseudomonas aeruginosa azurin to determine the relative spectral contribution from each of the copper ligands. Growth on WS-sulfate labels the cysteine ligand and allows the identification of a cluster of bands with Cu-S(Cys) stretching character between 370 and 430 cm(-1) whose frequencies are consistent with the trigonal or distorted tetrahedral coordination in type 1 sites. In type 2 copper-cysteinate sites, the lower nu(Cu-S) frequencies between 260 and 320 cm(-1) are consistent with square-planar coordination. Addition of exogenous N-15-labeled imidazole or histidine to the His117Gly mutant generates type 1 or type 2 sites, respectively. Because neither the above nor the His46Gly mutant reconstituted with N-15-imidazole exhibits significant isotope dependence, the histidine ligands can be ruled out as important contributors to the RR spectrum. Instead, a variety of evidence, including extensive isotope shifts upon global substitution with N-15, suggests that the multiple RR modes of azurin are due principally to vibrations of the cysteine ligand. These are resonance-enhanced through kinematic coupling with the Cu-S stretch in the ground state or through an excited-state A-term mechanism involving a Cu-cysteinate chromophore that extends into the peptide backbone.