Biological applications of resonance Raman spectroscopy: Structural characterization of copper-cysteinate active sites in blue copper proteins

This dissertation aims to further increase our knowledge about molecular mechanisms in proteins containing transition metal ions at their active sites by utilizing the sensitivity and selectivity of resonance Raman (RR) scattering as a probe of structure and bonding. The principal targets are biochemical structures operating at the mononuclear copper-cysteinate sites of blue copper proteins, the ubiquitous electron-transfer agents in nature. Specifically, the structural effects due to (1) the transition metal ion, (2) the nature of the axial ligands, (3) constrained protein environments, (4) hydrogen bond networks, (5) the hydrophobicity of the metal binding pocket, (6) weak pi-pi stacking interactions, and (7) the protein reduction potentials are elucidated. Using RR spectroscopy at cryogenic temperature and site-directed mutagenesis, the role of axial methionine 121 in modulating the reduction potential of the copper(II) ion has been investigated with a series of Pseudomonas aeruginosa azurin mutants containing isostructural unnatural amino acid analogues. Linear correlations were found between the Cu(II)-S(Cys) stretching vibrations and both the reduction potential and the hydrophobicity of the variants. A comparative RR study of azurins I and II from the same bacterium Alcaligenes xylosoxidans showed that the protein-induced structural differences at the copper site are due to the altered amino acid compositions in the Cys112-His117 and His117-Met121 loops. Structural interpretation of the RR spectrum of nickel(II)-substituted A. xylosoxidans azurin I revealed substantial weakening of the M(II)-S(Cys) bond upon replacement of Cu(II) with Ni(II), which is attributed to the combined effects of structural distortion toward tetrahedral coordination and the smaller effective nuclear charge of the divalent nickel compared to the trigonal copper(II). The effects of D2O/H2O exchange on the RR spectra of green alga Ulva pertusa and fern Dryopteris crassirhizoma plastocyanins provided useful vibrational markers for plastocyanin structural alterations and showed selective NH/D exchange on the water-exposed imidazole rings of histidine ligands. Finally, the question of weak pi-pi stacking interactions between the copper ligated histidines and the remote aromatic side chains in blue copper proteins is examined using a series of Achromobacter cycloclastes pseudoazurin mutated at the methionine 16 position with aromatic (Tyr, Trp, and Phe) and aliphatic (Ala, Val, Leu, or Ile) amino acids.