title: Cell-free synthesis of cytochrome c oxidase, a multicomponent membrane protein.
abstract: 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.
authors.affiliation: Picobiology Institute, Graduate School of Life Science, Center of Advanced Science and Technology, University of Hyogo, 3-1-1 Koto, Kamighori, Akoh, Hyogo, 678-1205, Japan.
authors.forename: Yukie
authors.initials: Y
authors.lastname: Katayama
authors.forename: Kunitoshi
authors.initials: K
authors.lastname: Shimokata
authors.forename: Makoto
authors.initials: M
authors.lastname: Suematsu
authors.forename: Takashi
authors.initials: T
authors.lastname: Ogura
authors.forename: Tomitake
authors.initials: T
authors.lastname: Tsukihara
authors.forename: Shinya
authors.initials: S
authors.lastname: Yoshikawa
authors.forename: Hideo
authors.initials: H
authors.lastname: Shimada
mesh_terms: D002474:Cell-Free System; D003001:Cloning, Molecular / Q000379:methods*; D045304:Cytochromes c / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000096:biosynthesis* / Q000138:chemical synthesis; D004926:Escherichia coli / Q000235:genetics; D008722:Methods; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000235:genetics
chemical_list: D045304:Cytochromes c; D003576:Electron Transport Complex IV