Biogenesis of the bacterial cbb3 cytochrome c oxidase: Active subcomplexes support a sequential assembly model

Abstract

The cbb₃ oxidase has a high affinity for oxygen and is required for growth of bacteria, including pathogens, in oxygen-limited environments. However, the assembly of this oxidase is poorly understood. Most cbb₃ are composed of four subunits: the catalytic CcoN subunit, the two cytochrome c subunits (CcoO and CcoP) involved in electron transfer, and the small CcoQ subunit with an unclear function. Here, we address the role of these four subunits in cbb₃ biogenesis in the purple bacterium Rubrivivax gelatinosus Analyses of membrane proteins from different mutants revealed the presence of active CcoNQO and CcoNO subcomplexes and also showed that the CcoP subunit is not essential for their assembly. However, CcoP was required for the oxygen reduction activity in the absence of CcoQ. We also found that CcoQ is dispensable for forming an active CcoNOP subcomplex in membranes. CcoNOP exhibited oxygen reductase activity, indicating that the cofactors (hemes b and copper for CcoN and cytochromes c for CcoO and CcoP) were present within the subunits. Finally, we discovered the presence of a CcoNQ subcomplex and showed that CcoN is the required anchor for the assembly of the full CcoNQOP complex. On the basis of these findings, we propose a sequential assembly model in which the CcoQ subunit is required for the early maturation step: CcoQ first associates with CcoN before the CcoNQ-CcoO interaction. CcoP associates to CcoNQO subcomplex in the late maturation step, and once the CcoNQOP complex is fully formed, CcoQ is released for degradation by the FtsH protease. This model could be conserved in other bacteria, including the pathogenic bacteria lacking the assembly factor CcoH as in R. gelatinosus.

Keywords: bacteria; cbb3 cytochrome c oxidase biogenesis; cytochrome oxidase; membrane protein; membrane sub-complexes assembly; protein assembly; respiration; subunit interactions.

Figure 1.

cbb₃-Cox expression and activity. A, oxygen consumption of WT, cbb₃⁻, and bd⁻. The WT was grown under aerobiosis (AE), semiaerobiosis (SA), or microaerobiosis (μA). cbb₃⁻ (ccoN::Km) and bd⁻ (cydA::Ω) mutants were grown under microaerobiosis. Non-inoculated medium was used as a control. B, cbb₃ oxidase in-gel activity staining of n-dodecyl β-d-maltopyranoside-solubilized membranes of WT grown under various conditions and cbb₃⁻ mutant grown under microaerobiosis. Activity was detected by 7.5–12% acrylamide gradient BN-PAGE in the presence of DAB. PS, photosynthesis. C, SDS-PAGE, Western blotting, and immunodetection of cbb₃ subunits using antibodies raised against each subunit. WT microaerobiosis- and semiaerobiosis-grown cells were harvested after 5 and 4 h at 0% O₂, respectively. Total membrane proteins were heated at 37 °C for 10 min, and 10 μg was loaded. Scatter plots demonstrating the quantitation (ImageJ) of activity or protein amount are shown. Results are the average of three to four different experiments. The quantification is shown relative to aerobic condition. The results are expressed as the mean ± S.E. (error bars). Significance of variation was determined by one-way ANOVA with Dunnett's multiple comparison test. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.1; ns, non-significant.

Figure 2.

Microaerobic expression profile of cbb₃ at different time points. A, cbb₃ oxidase in-gel activity staining after BN-PAGE analysis. B, BN-PAGE, Western blotting, and immunodetection of cbb₃ subunits. For the detection of CcoN, the membrane was first probed with anti-CcoQ antibodies, then stripped, and reprobed with anti-CcoN antibodies. C, SDS-PAGE, Western blotting, and immunodetection of cbb₃ subunits. 10 μg of protein was loaded except for the membrane revealed with anti-CcoQ for which only 3.3 μg of total membranes was loaded. Membrane proteins from cbb₃ mutants were loaded as a negative control as indicated. Quantification of cbb₃ subunits levels detected by immunoblotting and showing the decrease of CcoQ was performed using ImageJ. The ratio was normalized relative to time point 0 h.

Figure 3.

CcoQ accumulation in ftsH deletion mutant. WT and ftsH⁻ cells were harvested at different times once the dissolved oxygen reached zero. A, cbb₃ oxidase in-gel activity staining. B, SDS-PAGE, Western blotting, and immunodetection of cbb₃ subunits. The membrane proteins of ccoN⁻ mutant were used as a control. Quantification of cbb₃ subunits levels detected by immunoblotting showing the increase of CcoQ in ftsH⁻ cells was performed using ImageJ. Results are the average of two different experiments. The quantification is shown as relative to time point 7 h. The results are expressed as the mean ± S.E. (error bars). C, BN-PAGE, Western blotting, and immunodetection of cbb₃. * indicates the low molecular weight band detected with the anti-CcoQ antibodies. LH-RC, light-harvesting reaction center.

Figure 4.

Importance of cbb₃ subunits for oxygen consumption. All the strains were cultivated under microaerobiosis conditions. A, oxygen consumption rates (percentage of dissolved oxygen/h). Results are the average of five different experiments. B, doubling time of WT and mutants under microaerobiosis. Results are the average of three to five different experiments. The results are expressed as the mean ± S.E. (error bars). Significance of variation was determined by one-way ANOVA with Dunnett's multiple comparison test. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01.

Figure 5.

Importance of cbb₃ subunits for a fully active and stable complex. All strains were cultivated under microaerobiosis conditions. WT membrane of cells harvested after 15 h at 0% O₂ was used as a control. A, cbb₃ oxidase in-gel activity staining after overnight reaction. B, SDS-PAGE, Western blotting, and immunodetection of cbb₃ subunits. C, BN-PAGE, Western blotting, and immunodetection of cbb₃ subcomplexes. The name of the subunits of the detected subcomplexes are colored according to the assembly model scheme (Fig. 7).

Figure 6.

Detection of NO and NQ subcomplexes. All the strains were cultivated under microaerobiosis conditions. A, SDS-PAGE, Western blotting, and immunodetection of cbb₃ subunits. B, cbb₃ oxidase in-gel activity staining. C, BN-PAGE, Western blotting, and immunodetection of cbb₃ subcomplexes. The name of the subunits of the detected subcomplexes are colored according to the assembly model scheme (Fig. 7).

Figure 7.

cbb₃-Cox assembly model and intermediate subcomplexes in the membrane. The model depicts the sequential assembly of cbb₃ and the intermediates formed during this process. CcoQ could stabilize CcoN. CcoO binds to the CcoNQ complex, allowing association of CcoP. CcoQ is unloaded and degraded once the complex is fully assembled. All the depicted subcomplexes were detected either in the WT or in the appropriate mutants. The heme and copper in the isolated CcoN have not been demonstrated but seem to be independent of CcoQ attachment. The stoichiometry of CcoQ is speculative at this stage. IM, inner membrane.