Discovery And Function Study Of CydX, A Third Subunit In Cytochrom Bd Oxidase From Shewanella Oneidensis

Cytochrome bd oxidase is a respiratory terminal oxidase existing extensively in prokaryotes especially in some bacterial pathogens. It becomes indispensable for some bacteria to thrive under microaerobic conditions given its extremely high affinity for oxygen. Additionally, cytochrome bd oxidase confers bacteria the ability to tolerate nitric oxide (NO), a common immune defense agent released by some host immune cells. Shewanella oneidensis MR-1, a Gram-negative facultative anaerobe inhabiting in watery environments, is famous for its exceptional ability to respire on a huge array of inorganic and organic electron acceptors. MR-1 has received considerable attention for its potential applications in bioremediation of heavy metals and energy generation via fuel cells. Cytochrome bd oxidase in MR-1 is the enzyme that helps the cell to tolerate nitrite which can serve as an electron acceptor and universally exists in environments.Deletion of a small ORF so3284 (cydX) immediately following cydAB, the coding genes for cytochrome bd oxidase, resulted in substantially reduced resistance of MR-1 to nitrite. Complementation of the deletion by this small gene and its homologous counterparts from E. coli was able to restore its ability to resist nitrite. We thus hypothesized that CydX is the third subunit of the cytochrome bd oxidase in S. oneidensis. To confirm, we found that on transcriptional level, cydX was co-transcribed with cydAB as evidenced by RT-PCR. Structurally, CydX was predicted to be a single-transmembrane protein. Our results demonstrated that CydX can interact with CydAB in the inner membrane, forming a complex, with its C terminal in the cytosol and N terminal in the periplasm.We showed here that CydX expression, targeting and integration to the membrane is independent of CydAB, while CydAB is able to form membrane complex regardless of CydX. Mutagenesis analysis showed that the conserved N terminal part, specifically, the transmembrane segment and the pentapeptide exposed in the periplasm is necessary for the complex to become fully functional. Surprisingly, single point mutation of the most conserved residues in CydX is not sufficient to cause a phenotypic defect in terms of nitrite resistance. Since no crystal structure for cytochrome bd oxidase has been reported so far and CydX only exists in a small group of bacteria, this study would be helpful for future effort to investigate structure and function of cytochrome bd oxidase along with the structural and functional differences between cytochrom bd oxidases with and without the subunit.