title: Reduction potential variations in azurin through secondary coordination sphere phenylalanine incorporations.
abstract: Recent evidence has shown that the properties of metal binding sites can be tuned by more than the ligands in the primary coordination sphere. We investigated the incorporation of four phenylalanine residues into the secondary coordination sphere of the small soluble blue copper protein azurin. The locations for placement of these residues in azurin were based on the structure of the highly hydrophobic blue copper protein rusticyanin, which is known to have a significantly higher reduction potential than azurin. Using site-directed mutagenesis, these residues in close proximity to the copper binding site were mutated to large hydrophobic phenylalanine residues individually and in combination. We also added the Met121Leu mutation on top of the Phe mutations to construct a total of 13 variants. We found little change in the UV-visible absorption and EPR data for these proteins, however modest increases in reduction potential were observed with increases by as much as 30mV per Phe residue. Furthermore, we observed the increases in potential to be additive.
authors.affiliation: Department of Chemistry and Biochemistry, University of Minnesota Duluth, Duluth, MN 55812, USA. smberry@d.umn.edu
authors.forename: Steven M
authors.initials: SM
authors.lastname: Berry
authors.forename: Madelyn H
authors.initials: MH
authors.lastname: Baker
authors.forename: Nicole J
authors.initials: NJ
authors.lastname: Reardon
mesh_terms: D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D055664:Electrochemical Techniques; D004578:Electron Spin Resonance Spectroscopy; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D013053:Spectrophotometry
chemical_list: C012543:rusticyanin; D001400:Azurin; D010649:Phenylalanine