Trapping intermediates in metal transfer reactions of the CusCBAF export pump of Escherichia coli

Kelly N. Chacón, Jonathan Perkins, Zachary Mathe, Katherine Alwan, Ethan N. Ho, Melek N. Ucisik, Kenneth M. Merz, Ninian J. Blackburn

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

Escherichia coli CusCBAF represents an important class of bacterial efflux pump exhibiting selectivity towards Cu(I) and Ag(I). The complex is comprised of three proteins: the CusA transmembrane pump, the CusB soluble adaptor protein, and the CusC outer-membrane pore, and additionally requires the periplasmic metallochaperone CusF. Here we used spectroscopic and kinetic tools to probe the mechanism of copper transfer between CusF and CusB using selenomethionine labeling of the metal-binding Met residues coupled to RFQ-XAS at the Se and Cu edges. The results indicate fast formation of a protein−protein complex followed by slower intra-complex metal transfer. An intermediate coordinated by ligands from each protein forms in 100 ms. Stopped-flow fluorescence of the capping CusF-W44 tryptophan that is quenched by metal transfer also supports this mechanism. The rate constants validate a process in which shared-ligand complex formation assists protein association, providing a driving force that raises the rate into the diffusion-limited regime.

Original languageEnglish (US)
Article number192
JournalCommunications Biology
Volume1
Issue number1
DOIs
StatePublished - Dec 1 2018

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • Medicine (miscellaneous)

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