Deutsch
 
Benutzerhandbuch Datenschutzhinweis Impressum Kontakt
  DetailsucheBrowse

Datensatz

DATENSATZ AKTIONENEXPORT

Freigegeben

Zeitschriftenartikel

Biochemical and Biophysical Characterization of the Two Isoforms of cbb3-Type Cytochrome c Oxidase from Pseudomonas stutzeri

MPG-Autoren
/persons/resource/persons137954

Xie,  Hao
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

/persons/resource/persons137616

Buschmann,  Sabine
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

/persons/resource/persons137770

Langer,  Julian D.
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

/persons/resource/persons137800

Michel,  Hartmut
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

Externe Ressourcen
Es sind keine Externen Ressourcen verfügbar
Volltexte (frei zugänglich)
Es sind keine frei zugänglichen Volltexte verfügbar
Ergänzendes Material (frei zugänglich)
Es sind keine frei zugänglichen Ergänzenden Materialien verfügbar
Zitation

Xie, H., Buschmann, S., Langer, J. D., Ludwig, B., & Michel, H. (2014). Biochemical and Biophysical Characterization of the Two Isoforms of cbb3-Type Cytochrome c Oxidase from Pseudomonas stutzeri. Journal of Bacteriology (Washington, DC), 196(2), 472-482. doi:10.1128/JB.01072-13.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0026-B19F-3
Zusammenfassung
The cbb3-type cytochrome c oxidases (cbb3-CcOs) are members of the heme-copper oxidase superfamily that couple the reduction of oxygen to translocation of protons across the membrane. The cbb3-CcOs are present only in bacteria and play a primary role in microaerobic respiration, being essential for nitrogen-fixing endosymbionts and for some human pathogens. As frequently observed in Pseudomonads, Pseudomonas stutzeri contains two independent ccoNO(Q)P operons encoding the two cbb3 isoforms, Cbb3-1 and Cbb3-2. While the crystal structure of Cbb3-1 from P. stutzeri was determined recently and cbb3-CcOs from other organisms were characterized functionally, less emphasis has been placed on the isoform-specific differences between the cbb3-CcOs. In this work, both isoforms were homologously expressed in P. stutzeri strains from which the genomic version of the respective operon was deleted. We purified both cbb3 isoforms separately by affinity chromatography and increased the yield of Cbb3-2 to a similar level as Cbb3-1 by replacing its native promoter. Mass spectrometry, UV-visible (UV-Vis) spectroscopy, differential scanning calorimetry, as well as oxygen reductase and catalase activity measurements were employed to characterize both cbb3 isoforms. Differences were found concerning the thermal stability and the presence of subunit CcoQ. However, no significant differences between the two isoforms were observed otherwise. Interestingly, a surprisingly high turnover of at least 2,000 electrons s-1 and a high Michaelis-Menten constant (Km-3.6 mM) using ascorbate–N,N,N',N'-tetramethyl-pphenylenediamine dihydrochloride (TMPD) as the electron donor were characteristic for both P. stutzeri cbb3-CcOs. Our work provides the basis for further mutagenesis studies of each of the two cbb3 isoforms specifically.