Abstract
For the first time, the functioning of the oxygen reductase Na+-pump (Na+-pum** cytochrome c oxidase of the cbb3-type) was demonstrated by examining the respiratory chain of the extremely alkaliphilic bacterium Thioalkalivibrio versutus [Muntyan, M. S., et al. (2015) Cytochrome cbb3 of Thioalkalivibrio is a Na+-pum** cytochrome oxidase, Proc. Natl. Acad. Sci. USA, 112, 7695-7700], a product of the ccoNOQP operon. In this study, we detected and identified this enzyme using rabbit polyclonal antibody against the predicted C-terminal amino acid sequence of its catalytic subunit. We found that this cbb3-type oxidase is synthesized in bacterial cells, where it is located in the membranes. The 48-kDa oxidase subunit (CcoN) is catalytic, while subunits CcoO and CcoP with molecular masses of 29 and 34 kDa, respectively, are cytochromes c. The theoretical pI values of the CcoN, CcoO, and CcoP subunits were determined. It was shown that parts of the CcoO and CcoP subunits exposed to the aqueous phase on the cytoplasmic membrane P-side are enriched with negatively charged amino acid residues, in contrast to the parts of the integral subunit CcoN adjacent to the aqueous phase. Thus, the Na+-pum** cytochrome c oxidase of T. versutus, both in function and in structure, demonstrates adaptation to extremely alkaline conditions.
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Abbreviations
- AS:
-
ammonium sulphate
- CO:
-
cytochrome c oxidase
- NQR:
-
Na+:NADH quinone oxidoreductase
- OR-ETC:
-
oxygen reductase electron-transporting chain
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Acknowledgements
The authors express their gratitude to V. P. Skulachev for constant interest and fruitful discussion of their research and to N. V. Pozdnyakova for the help in the work with animals.
Funding
This work was supported by the Russian Foundation for Basic Research (projects nos. 02-04-49107-a and 05-04-49504-a, genome studies; project no. 20-04-01105-a, protein studies).
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The authors declare no conflict of interest in financial or any other sphere. The handling of the rabbits and their use in the experiments were in compliance with the protocols developed and approved by the Animal Ethics Committee of the Belozersky Institute of Physico-Chemical Biology. All procedures were in accordance with the guidelines of the Federation of Laboratory Animal Science Associations (FELASA).
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Muntyan, M.S., Morozov, D.A., Leonova, Y.F. et al. Identification of Na+-Pum** Cytochrome Oxidase in the Membranes of Extremely Alkaliphilic Thioalkalivibrio Bacteria. Biochemistry Moscow 85, 1631–1639 (2020). https://doi.org/10.1134/S0006297920120147
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DOI: https://doi.org/10.1134/S0006297920120147