Mechanism of proton transfer through the KC proton pathway in the Vibrio cholerae cbb3 terminal oxidase

Young O. Ahn, Ingrid Albertsson, Robert B. Gennis, Pia Ädelroth

Research output: Contribution to journalArticle

Abstract

The heme‑copper oxidases (HCuOs) are terminal components of the respiratory chain, catalyzing oxygen reduction coupled to the generation of a proton motive force. The C-family HCuOs, found in many pathogenic bacteria under low oxygen tension, utilize a single proton uptake pathway to deliver protons both for O2 reduction and for proton pumping. This pathway, called the KC-pathway, starts at Glu-49P in the accessory subunit CcoP, and connects into the catalytic subunit CcoN via the polar residues Tyr-(Y)-227, Asn (N)-293, Ser (S)-244, Tyr (Y)-321 and internal water molecules, and continues to the active site. However, although the residues are known to be functionally important, little is known about the mechanism and dynamics of proton transfer in the KC-pathway. Here, we studied variants of Y227, N293 and Y321. Our results show that in the N293L variant, proton-coupled electron transfer is slowed during single-turnover oxygen reduction, and moreover it shows a pH dependence that is not observed in wildtype. This suggests that there is a shift in the pKa of an internal proton donor into an experimentally accessible range, from >10 in wildtype to ~8.8 in N293L. Furthermore, we show that there are distinct roles for the conserved Y321 and Y227. In Y321F, proton uptake from bulk solution is greatly impaired, whereas Y227F shows wildtype-like rates and retains ~50% turnover activity. These tyrosines have evolutionary counterparts in the K-pathway of B-family HCuOs, but they do not have the same roles, indicating diversity in the proton transfer dynamics in the HCuO superfamily.

Original languageEnglish (US)
Pages (from-to)1191-1198
Number of pages8
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1859
Issue number11
DOIs
StatePublished - Nov 2018

Keywords

  • Bioenergetics
  • Electron transfer
  • Liposomes
  • Oxygen reduction
  • Proton pumping

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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