Evidence for the requirement of CydX in function but not assembly of the cytochrome bd oxidase in Shewanella oneidensis

Background: Cytochrome bd oxidase, existing widely in bacteria, produces a proton motive force by the vectorial charge transfer of protons and more importantly, endows bacteria with a number of vitally important physiological functions, such as enhancing tolerance to various stresses. Although extensively studied as a CydA-CydB two-subunit complex for decades, the complex in certain groups of bacteria is recently found to in fact consist of an additional subunit, which is functionally essential. Methods: We investigated the assembly of the CydA-CydB complex using BiFC. We investigated the function of CydX using mutational analysis. Results: CydX, a 38-amino-acid inner-membrane protein, is associated with the CydA-CydB complex in Shewanella oneidensis, a facultative anaerobe renowned for its respiratory versatility. It is clear that CydX is neither required for the in vivo assembly of the CydA-CydB complex nor relies on the complex for its translocation and integration into the membrane. The N-terminal segment (1-25 amino acid residues) and short periplasmic overhang of CydX, with respect to functionality, are important whereas the remaining C-terminal segment is rather flexible. Conclusion: Based on these findings, we postulate that CydX may function by positioning and stabilizing the prosthetic hemes, especially heme din the CydA-CydB complex although a role of participating in catalytic reaction is not excluded. General significance: The work provides novel insights into our understanding of the small subunit of the cytochrome bd oxidase. (C) 2014 Elsevier B.V. All rights reserved.