Incorporation of the red copper nitrosocyanin binding loop into blue copper azurin

Steven M. Berry, Erika L. Bladholm, Elise J. Mostad, Audrey R. Schenewerk

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

Loop-directed mutagenesis was applied to the blue copper protein azurin to replace its copper binding loop with that from the red copper protein nitrosocyanin. A ten amino acid long loop that provides three of the four copper ligands from nitrosocyanin was incorporated into azurin to make a variant called NC-azurin. The chimeric protein displayed a red color, and UV-vis absorption and EPR spectra that closely resembled those of the loop parent, nitrosocyanin. We added the fourth ligand from nitrosocyanin into NC-azurin, a carboxylate-containing amino acid, but the proteins had altered stability and spectroscopic properties that did not resemble those of either parent copper protein. The loop alone, however, was enough to impart red copper site characteristics to the NC-azurin protein. Finally, the reduction potential of the variant was found to be between the reduction potentials of the parent proteins and about 50 mV below that of wildtype azurin.

Original languageEnglish (US)
Pages (from-to)473-480
Number of pages8
JournalJournal of Biological Inorganic Chemistry
Volume16
Issue number3
DOIs
StatePublished - Mar 1 2011

Keywords

  • Azurin
  • Blue copper protein
  • Loop-directed mutagenesis
  • Nitrosocyanin
  • Red copper protein

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