title: Identification and Characterization of the Novel Subunit CcoM in the cbb3₃Cytochrome c Oxidase from Pseudomonas stutzeri ZoBell.
abstract: Cytochrome c oxidases (CcOs), members of the heme-copper containing oxidase (HCO) superfamily, are the terminal enzymes of aerobic respiratory chains. The cbb3-type cytochrome c oxidases (cbb3-CcO) form the C-family and have only the central catalytic subunit in common with the A- and B-family HCOs. In Pseudomonas stutzeri, two cbb3 operons are organized in a tandem repeat. The atomic structure of the first cbb3 isoform (Cbb3-1) was determined at 3.2 Å resolution in 2010 (S. Buschmann, E. Warkentin, H. Xie, J. D. Langer, U. Ermler, and H. Michel, Science 329:327-330, 2010, http://dx.doi.org/10.1126/science.1187303). Unexpectedly, the electron density map of Cbb3-1 revealed the presence of an additional transmembrane helix (TMH) which could not be assigned to any known protein. We now identified this TMH as the previously uncharacterized protein PstZoBell_05036, using a customized matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry setup. The amino acid sequence matches the electron density of the unassigned TMH. Consequently, the protein was renamed CcoM. In order to identify the function of this new subunit in the cbb3 complex, we generated and analyzed a CcoM knockout strain. The results of the biochemical and biophysical characterization indicate that CcoM may be involved in CcO complex assembly or stabilization. In addition, we found that CcoM plays a role in anaerobic respiration, as the ΔCcoM strain displayed altered growth rates under anaerobic denitrifying conditions.
  
  
  OBJECTIVE
  The respiratory chain has recently moved into the focus for drug development against prokaryotic human pathogens, in particular, for multiresistant strains (P. Murima, J. D. McKinney, and K. Pethe, Chem Biol 21:1423-1432, 2014, http://dx.doi.org/10.1016/j.chembiol.2014.08.020). cbb3-CcO is an essential enzyme for many different pathogenic bacterial species, e.g., Helicobacter pylori, Vibrio cholerae, and Pseudomonas aeruginosa, and represents a promising drug target. In order to develop compounds targeting these proteins, a detailed understanding of the molecular architecture and function is required. Here we identified and characterized a novel subunit, CcoM, in the cbb3-CcO complex and thereby completed the crystal structure of the Cbb3 oxidase from Pseudomonas stutzeri, a bacterium closely related to the human pathogen Pseudomonas aeruginosa.
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Kohlstaedt
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Buschmann
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
authors.forename: Hao
authors.initials: H
authors.lastname: Xie
authors.affiliation: Bruker Daltonik GmbH, Bremen, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Resemann
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
authors.forename: Eberhard
authors.initials: E
authors.lastname: Warkentin
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany julian.langer@biophys.mpg.de hartmut.michel@biophys.mpg.de.
authors.forename: Julian D
authors.initials: JD
authors.lastname: Langer
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany julian.langer@biophys.mpg.de hartmut.michel@biophys.mpg.de.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Michel
mesh_terms: D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055786:Gene Knockout Techniques; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D044223:Pseudomonas stutzeri / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D021122:Protein Subunits; C414443:cbb3 oxidase; D003576:Electron Transport Complex IV
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