The heme-copper oxidases (HCOs) accomplish the key event of aerobic respiration; they couple O2 reduction and transmembrane proton pumping. To gain new insights into the still enigmatic process, we structurally characterized a C-family HCO--essential for the pathogenicity of many bacteria--that differs from the two other HCO families, A and B, that have been structurally analyzed. The x-ray structure of the C-family cbb3 oxidase from Pseudomonas stutzeri at 3.2 angstrom resolution shows an electron supply system different from families A and B. Like family-B HCOs, C HCOs have only one pathway, which conducts protons via an alternative tyrosine-histidine cross-link. Structural differences around hemes b and b3 suggest a different redox-driven proton-pumping mechanism and provide clues to explain the higher activity of family-C HCOs at low oxygen concentrations.

The treatment of infectious diseases caused by multidrug-resistant pathogens is a major clinical challenge of the 21st century. The membrane-embedded respiratory cytochrome bd-type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. Escherichia coli encodes for two cytochrome bd isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between cydABX (Ecbd-I) and appCBX (Ecbd-II) operon encoded cytochrome bd variants have remained elusive in the past. Here, we demonstrate that cytochrome bd-II catalyzes oxidation of benzoquinols while possessing additional specificity for naphthoquinones. Our data show that although menaquinol-1 (MK1) is not able to directly transfer electrons onto cytochrome bd-II from E. coli, it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome bd-II to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two bd oxidase isoforms, but two notable differences are apparent upon inspection: (i) Ecbd-II does not contain a CydH-like subunit, thereby exposing heme b595 to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of Ecbd-I. Our work completes the structural landscape of terminal respiratory oxygen reductases of E. coli and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in E. coli.

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.

The cbb 3 -type cytochrome c oxidases (cbb 3 -CcOs) are members of the heme-copper oxidase superfamily that couple the reduction of oxygen to translocation of protons across the membrane. The cbb 3 -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 cbb 3 isoforms, Cbb 3 -1 and Cbb 3 -2. While the crystal structure of Cbb 3 -1 from P. stutzeri was determined recently and cbb 3 -CcOs from other organisms were characterized functionally, less emphasis has been placed on the isoform-specific differences between the cbb 3 -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 cbb 3 isoforms separately by affinity chromatography and increased the yield of Cbb 3 -2 to a similar level as Cbb 3 -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 cbb 3 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 (K m ϳ 3.6 mM) using ascorbate-N,N,N=,N=-tetramethyl-pphenylenediamine dihydrochloride (TMPD) as the electron donor were characteristic for both P. stutzeri cbb 3 -CcOs. Our work provides the basis for further mutagenesis studies of each of the two cbb 3 isoforms specifically.

The Cbb-type cytochrome c oxidases (Cbb-CcOs), the second most abundant CcOs, catalyze the reduction of molecular oxygen to water, even at micromolar oxygen concentrations. In Pseudomonas stutzeri ZoBell, two tandemly organized cbb-operons encode the isoforms Cbb-1 and Cbb-2 both possessing subunits CcoN, CcoO and CcoP. However, only the cbb-2 operon contains an additional ccoQ gene. CcoQ consists of 62 amino acids and is predicted to possess one transmembrane spanning helix. The physiological role of CcoQ was investigated based on a CcoQ-deletion mutant and wild-type Cbb-2 crystals not containing subunit CcoQ. Cbb-2 isolated from the deletion mutant is inactive and appears as a dispersed band on blue native-PAGE gels. Surprisingly, in the absence of ccoQ, Cbb-1 also shows a strongly reduced activity. Our data suggest that CcoQ primarily functions as an assembly factor for Cbb-2 but is also required for correct assembly of Cbb-1. In contrast, once correctly assembled, Cbb-1 and Cbb-2 possess a full enzymatic activity even in the absence of CcoQ.