Non-covalent interactions in blue copper protein probed by Met16 mutation and electronic and resonance Raman spectroscopy of Achromobacter cycloclastes pseudoazurin

We have used low-temperature (77 K) resonance Raman (RR) spectroscopy as a probe of the electronic and molecular structure to investigate weak π–π interactions between the metal ion-coordinated His imidazoles and aromatic side chains in the second coordination sphere of blue copper proteins. For this purpose, the RR spectra of Met16 mutants of Achromobacter cycloclastes pseudoazurin (AcPAz) with aromatic (Met16Tyr, Met16Trp, and Met16Phe) and aliphatic (Met16Ala, Met16Val, Met16Leu, and Met16Ile) amino acid side chains have been obtained and analyzed over the 100–500 cm−1 spectral region. Subtle strengthening of the Cu(II)–S(Cys) interaction on replacing Met16 with Tyr, Trp, and Phe is indicated by the upshifted (0.3–0.8 cm−1) RR bands involving ν(Cu–S)Cys stretching modes. In contrast, the RR spectra of Met16 mutants with aliphatic amino acids revealed larger (0.2–1.8 cm−1) shifts of the ν(Cu–S)Cys stretching modes to a lower frequency region, which indicate a weakening of the Cu(II)–S(Cys) bond. Comparisons of the predominantly ν(Cu–S)Cys stretching RR peaks of the Met16X = Tyr, Trp, and Phe variants, with the molar absorptivity ratio ε1/ε2 of σ(∼455 nm)/π(∼595 nm) (Cys)S → Cu(II) charge-transfer bands in the optical spectrum and the axial/rhombic EPR signals, revealed a slightly more trigonal disposition of ligands about the copper(II) ion. In contrast, the RR spectra of Met16Z = Ala, Val, Leu, and Ile variants with aliphatic amino acid side chains show a more tetrahedral perturbation of the copper active site, as judged by the lower frequencies of the ν(Cu–S)Cys stretching modes, much larger values of the ε1/ε2 ratio, and the increased rhombicity of the EPR spectra.