Designing novel blue copper and purple CuA centers in azurin with natural and unnatural amino acids.

摘要

The goal of this work is to develop new aspects of metalloprotein design with a focus on the equatorial His ligands of the dinuclear Cu_A site, the Cys and axial Met ligands of the type 1 copper site of azurin, and the design of a dinuclear iron site. We chose to study the properties of the Cu_A site through the designed Cu_A azurin model. We probed the role of His120 in Cu_A azurin by mutating it to Asp, Asn, Ala, and Gly. UV-visible absorption, CD, MCD, resonance Raman, EXAFS, and ENDOR data of the mutants strongly resembled the parent CuA azurin spectra and suggested that the mutants resembled all valence delocalized Cu_A centers. However, four line EPR spectra of the mutants implied that the site was valence trapped. Possible replacement ligands supporting the dinuclear site were ruled out by amino acid mutations and titrations with exogenous ligands.; A limitation of the site-directed mutagenesis approach is the availability of only 20 amino acids naturally occurring in the genetic code. The highly conserved methionine ligand was replaced with the unnatural and isostructural amino acids norleucine (Nle) and selenomethionine (SeM) in azurin using expressed protein ligation (EPL). An analysis of the variants revealed hydrophobicity as the dominant factor in tuning the reduction potentials of blue copper centers by axial ligands. Also using EPL, an azurin variant containing selenocysteine in place of the cysteine ligand was synthesized. The variant displayed spectroscopic properties that were consistent with a decrease in covalency of the Cu-Se bond, compared to the Cu-S bond. The preparation of a mixed selenolate selenoether complex through the EPL synthesis of SeMet121 SeCys112 azurin was prepared to complement the study of SeMet azurin. These studies illustrate that iso-structural substitution using EPL can fine-tune the structural and functional properties of a metal-binding site without loss of its characteristics or metal binding properties.; Finally, loop-directed mutagenesis was used to incorporate a 2Fe-2S cluster binding loop from human ferrochelatase into azurin. Preliminary iron binding studies indicate the assembly of a 4Fe-4S or a 2Fe-2S iron sulfur cluster.