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Media type:
E-Article
Title:
Mutation of a single residue in theba₃ oxidase specifically impairs protonation of the pump site
Contributor:
von Ballmoos, Christoph;
Gonska, Nathalie;
Lachmann, Peter;
Gennis, Robert B.;
Ädelroth, Pia;
Brzezinski, Peter
imprint:
National Academy of Sciences, 2015
Published in:Proceedings of the National Academy of Sciences of the United States of America
Language:
English
ISSN:
0027-8424;
1091-6490
Origination:
Footnote:
Description:
<p>The<italic>ba</italic>₃-type cytochrome<italic>c</italic>oxidase from<italic>Thermus thermophilus</italic>is a membrane-bound protein complex that couples electron transfer to O₂ to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the<italic>ba</italic>₃ oxidase where a putative “pump site” was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O₂ reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome<italic>c</italic>oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome<italic>c</italic>oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the<italic>ba</italic>₃ cytochrome<italic>c</italic>oxidase.</p>