Copper binding and reactivity at the histidine brace motif: insights from mutational analysis of the Pseudomonas fluorescens copper chaperone CopC

Johan Ø Ipsen, Cristina Hernández-Rollán, Martin Muderspach Thellefsen, Søren Brander, Andreas B Bertelsen, Poul Erik Jensen, Morten H H Nørholm, Leila Lo Leggio, Katja S Johansen

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

The histidine brace (His-brace) is a copper-binding motif that is associated with both oxidative enzymes and proteinaceous copper chaperones. Here, we used biochemical and structural methods to characterize mutants of a His-brace-containing copper chaperone from Pseudomonas fluorescens (PfCopC). A total of 15 amino acid variants in primary and second-sphere residues were produced and characterized in terms of their copper binding and redox properties. PfCopC has a very high affinity for Cu(II) and also binds Cu(I). A high reorganization barrier likely prevents redox cycling and, thus, catalysis. In contrast, mutations in the conserved second-sphere Glu27 enable slow oxidation of ascorbate. The crystal structure of the variant E27A confirmed copper binding at the His-brace. Unexpectedly, Asp83 at the equatorial position was shown to be indispensable for Cu(II) binding in the His-brace of PfCopC. A PfCopC mutant that was designed to mimic the His-brace from lytic polysaccharide monooxygenase-like family X325 did not bind Cu(II), but was still able to bind Cu(I). These results highlight the importance of the proteinaceous environment around the copper His-brace for reactivity and, thus, the difference between enzyme and chaperone.
Original languageEnglish
JournalFEBS Letters
Volume595
Issue number12
Pages (from-to)1708-1720
Number of pages13
ISSN0014-5793
DOIs
Publication statusPublished - 2021

Keywords

  • Ascorbate assay
  • Isothermal calorimetry
  • Protein
  • X-ray crystal structure

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