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 language | English (US) |
|---|---|
| Pages (from-to) | 1191-1198 |
| Number of pages | 8 |
| Journal | Biochimica et Biophysica Acta - Bioenergetics |
| Volume | 1859 |
| Issue number | 11 |
| DOIs | |
| State | Published - Nov 2018 |
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Keywords
- Bioenergetics
- Electron transfer
- Liposomes
- Oxygen reduction
- Proton pumping
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Cell Biology
Cite this
Mechanism of proton transfer through the KC proton pathway in the Vibrio cholerae cbb3 terminal oxidase. / Ahn, Young O.; Albertsson, Ingrid; Gennis, Robert B.; Ädelroth, Pia.
In: Biochimica et Biophysica Acta - Bioenergetics, Vol. 1859, No. 11, 11.2018, p. 1191-1198.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Mechanism of proton transfer through the KC proton pathway in the Vibrio cholerae cbb3 terminal oxidase
AU - Ahn, Young O.
AU - Albertsson, Ingrid
AU - Gennis, Robert B.
AU - Ädelroth, Pia
PY - 2018/11
Y1 - 2018/11
N2 - 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.
AB - 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.
KW - Bioenergetics
KW - Electron transfer
KW - Liposomes
KW - Oxygen reduction
KW - Proton pumping
UR - http://www.scopus.com/inward/record.url?scp=85052496601&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85052496601&partnerID=8YFLogxK
U2 - 10.1016/j.bbabio.2018.08.002
DO - 10.1016/j.bbabio.2018.08.002
M3 - Article
C2 - 30251700
AN - SCOPUS:85052496601
VL - 1859
SP - 1191
EP - 1198
JO - Biochimica et Biophysica Acta - Bioenergetics
JF - Biochimica et Biophysica Acta - Bioenergetics
SN - 0005-2728
IS - 11
ER -