Reduction potential variations in azurin through secondary coordination sphere phenylalanine incorporations

Steven M. Berry, Madelyn H. Baker, Nicole J. Reardon

Research output: Contribution to journalArticlepeer-review

33 Scopus citations


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 30. mV per Phe residue. Furthermore, we observed the increases in potential to be additive.

Original languageEnglish (US)
Pages (from-to)1071-1078
Number of pages8
JournalJournal of Inorganic Biochemistry
Issue number10
StatePublished - Oct 2010

Bibliographical note

Funding Information:
The authors thank the Swenson Family Foundation , UM-GIA , and the Camille and Henry Dreyfus Foundation for funding and student stipends. We thank Mark J. Nilges of the Illinois EPR Research Center for assistance in gathering EPR data. Finally, we would like to thank John H. Richards, California Institute of Technology and Yi Lu of the University of Illinois U.-C. for the kind gift of the azurin gene.


  • Azurin
  • Blue copper
  • Electrochemistry
  • Reduction potential
  • Secondary coordination sphere
  • Type 1 copper


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