Cell-free synthesis of cytochrome c oxidase, a multicomponent membrane protein

J Bioenerg Biomembr. 2010 Jun;42(3):235-40. doi: 10.1007/s10863-010-9285-8. Epub 2010 Apr 6.


Cell-free protein synthesis is a useful technique that can site-specifically incorporate isotope-labeled amino acids into proteins. This incorporation is essential for infrared analyses of the electronic state of a specific amino acid residue used to elucidate protein function. Although 17 membrane proteins have been synthesized in their active state by cell-free systems, to date no hetero-subunit protein has been synthesized with this technique, suggesting that there are serious technical limitations. Here we report the cell-free synthesis of Paracoccus denitrificans cytochrome c oxidase, a membrane protein complex composed of three distinct subunits that contain two heme A molecules and two redox-active copper centers. The synthesized protein exhibited normal Soret/vis absorption spectra and ferrocytochrome c oxidation activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell-Free System*
  • Cloning, Molecular / methods*
  • Cytochromes c / metabolism
  • Electron Transport Complex IV / biosynthesis*
  • Electron Transport Complex IV / chemical synthesis
  • Escherichia coli / genetics
  • Methods
  • Oxidation-Reduction
  • Paracoccus denitrificans / genetics


  • Cytochromes c
  • Electron Transport Complex IV