Identification of Na⁺-Pumping Cytochrome Oxidase in the Membranes of Extremely Alkaliphilic Thioalkalivibrio Bacteria

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M. S. Muntyan^1,a*, D. A. Morozov¹, Y. F. Leonova², and T. V. Ovchinnikova²

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

²Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

^* To whom correspondence should be addressed.

Received July 26, 2020; Revised September 1, 2020; Accepted September 1, 2020

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For the first time, the functioning of the oxygen reductase Na⁺-pump (Na⁺-pumping 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⁺-pumping 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⁺-pumping cytochrome c oxidase of T. versutus, both in function and in structure, demonstrates adaptation to extremely alkaline conditions.

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KEY WORDS: Na⁺-pump, cytochrome c oxidase, extremophily, alkaliphily, cbb₃-type oxidase

DOI: 10.1134/S0006297920120147

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