Identification and Characterization of the Novel Subunit CcoM in the cbb 3 -Cytochrome c Oxidase from Pseudomonas stutzeri ZoBell

Abstract

ABSTRACT Cytochrome c oxidases (C c Os), members of the heme-copper containing oxidase (HCO) superfamily, are the terminal enzymes of aerobic respiratory chains. The cbb 3 -type cytochrome c oxidases ( cbb 3 -C c O) form the C-family and have only the central catalytic subunit in common with the A- and B-family HCOs. In Pseudomonas stutzeri , two cbb 3 operons are organized in a tandem repeat. The atomic structure of the first cbb 3 isoform (Cbb 3 -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 Cbb 3 -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 cbb 3 complex, we generated and analyzed a CcoM knockout strain. The results of the biochemical and biophysical characterization indicate that CcoM may be involved in C c O 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. IMPORTANCE 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 ). cbb 3 -C c O 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 cbb 3 -C c O complex and thereby completed the crystal structure of the Cbb 3 oxidase from Pseudomonas stutzeri , a bacterium closely related to the human pathogen Pseudomonas aeruginosa .