Multi-step assembly pathway of the cbb3-type cytochrome c oxidase complex

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Cbb3-type cytochrome c oxidases are crucial for microaerobic respiration in proteobacteria.
• Their biogenesis, encoded by the ccoNOQP operon, relies on ccoGHIS gene products for cofactor insertion and enzyme maturation.

Purpose of the Study:

• To investigate the assembly pathway of cbb3-type cytochrome c oxidase in Rhodobacter capsulatus.
• To elucidate the roles of CcoGHIS proteins and individual subunits in complex formation and activity.

Main Methods:

• Blue-native polyacrylamide gel electrophoresis (BN-PAGE) was used to analyze protein complex formation.
• Genetic deletions of specific genes (ccoGHIS, ccoI, ccoH, ccoS) were employed to study their impact on assembly.

Main Results:

• A stable, inactive 210 kDa sub-complex (CcoNOQHS) was identified, which converts to an active 230 kDa CcoNOQP complex upon CcoP binding.
• Deletion of ccoGHIS severely impaired complex formation and CcoP stability.
• CcoH is essential for complex formation, while CcoS appears involved in a late maturation step.
• Heme cofactor insertion into CcoP and CcoO occurs before sub-complex formation, with CcoN cofactor insertion occurring at various stages.

Conclusions:

• A stepwise assembly model is proposed: CcoN, O, Q form an initial complex stabilized by CcoH and CcoS.
• Subsequent CcoP binding and dissociation of CcoH/CcoS yield the active CcoNOQP complex.
• The study clarifies the essential roles of accessory proteins in the functional assembly of this vital respiratory enzyme.