Abstract
Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidase, one such pathway starts near a conserved Asp-132 residue. Results from earlier studies have shown that replacement of Asp-132 by, e.g., Asn, slows proton uptake by a factor of ~5,000. Here, we show that proton uptake at full speed (~104s-1) can be restored in the Asp-132-Asn oxidase upon introduction of a second structural modification further inside the pathway (Asn-139-Thr) without compensating for the loss of the negative charge. This protonuptake rate was insensitive to Zn2+ addition, which in the wildtype cytochrome c oxidase slows the reaction, indicating that Asp-132 is required for Zn2+ binding. Furthermore, in the absence of Asp-132 and with Thr at position 139, at high pH (>9), proton uptake was significantly accelerated. Thus, the data indicate that Asp-132 is not strictly required for maintaining rapid proton uptake. Furthermore, despite the rapid proton uptake in the Asn-139-Thr/ Asp-132-Asn mutant cytochrome c oxidase, proton pumping was impaired, which indicates that the segment around these residues is functionally linked to pumping.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 8912-8917 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 110 |
| Issue number | 22 |
| DOIs | |
| State | Published - May 28 2013 |
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Keywords
- Cytochrome aa3
- Electron transfer
- Metalloprotein
- Respiration
- Transporter
ASJC Scopus subject areas
- General
Cite this
Role of aspartate 132 at the orifice of a proton pathway in cytochrome c oxidase. / Johansson, Ann Louise; Högbom, Martin; Carlsson, Jens; Gennis, Robert B; Brzezinski, Peter.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, No. 22, 28.05.2013, p. 8912-8917.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Role of aspartate 132 at the orifice of a proton pathway in cytochrome c oxidase
AU - Johansson, Ann Louise
AU - Högbom, Martin
AU - Carlsson, Jens
AU - Gennis, Robert B
AU - Brzezinski, Peter
PY - 2013/5/28
Y1 - 2013/5/28
N2 - Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidase, one such pathway starts near a conserved Asp-132 residue. Results from earlier studies have shown that replacement of Asp-132 by, e.g., Asn, slows proton uptake by a factor of ~5,000. Here, we show that proton uptake at full speed (~104s-1) can be restored in the Asp-132-Asn oxidase upon introduction of a second structural modification further inside the pathway (Asn-139-Thr) without compensating for the loss of the negative charge. This protonuptake rate was insensitive to Zn2+ addition, which in the wildtype cytochrome c oxidase slows the reaction, indicating that Asp-132 is required for Zn2+ binding. Furthermore, in the absence of Asp-132 and with Thr at position 139, at high pH (>9), proton uptake was significantly accelerated. Thus, the data indicate that Asp-132 is not strictly required for maintaining rapid proton uptake. Furthermore, despite the rapid proton uptake in the Asn-139-Thr/ Asp-132-Asn mutant cytochrome c oxidase, proton pumping was impaired, which indicates that the segment around these residues is functionally linked to pumping.
AB - Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidase, one such pathway starts near a conserved Asp-132 residue. Results from earlier studies have shown that replacement of Asp-132 by, e.g., Asn, slows proton uptake by a factor of ~5,000. Here, we show that proton uptake at full speed (~104s-1) can be restored in the Asp-132-Asn oxidase upon introduction of a second structural modification further inside the pathway (Asn-139-Thr) without compensating for the loss of the negative charge. This protonuptake rate was insensitive to Zn2+ addition, which in the wildtype cytochrome c oxidase slows the reaction, indicating that Asp-132 is required for Zn2+ binding. Furthermore, in the absence of Asp-132 and with Thr at position 139, at high pH (>9), proton uptake was significantly accelerated. Thus, the data indicate that Asp-132 is not strictly required for maintaining rapid proton uptake. Furthermore, despite the rapid proton uptake in the Asn-139-Thr/ Asp-132-Asn mutant cytochrome c oxidase, proton pumping was impaired, which indicates that the segment around these residues is functionally linked to pumping.
KW - Cytochrome aa3
KW - Electron transfer
KW - Metalloprotein
KW - Respiration
KW - Transporter
UR - http://www.scopus.com/inward/record.url?scp=84878440015&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84878440015&partnerID=8YFLogxK
U2 - 10.1073/pnas.1303954110
DO - 10.1073/pnas.1303954110
M3 - Article
C2 - 23674679
AN - SCOPUS:84878440015
VL - 110
SP - 8912
EP - 8917
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 22
ER -