Mutation of a single residue in the ba 3 oxidase specifically impairs protonation of the pump site

• Published in: Proceedings of the National Academy of Sciences - PNAS 2015-03, Vol.112 (11), p.3397-3402
• Main Authors: von Ballmoos, Christoph, Gonska, Nathalie, Lachmann, Peter, Gennis, Robert B., Ädelroth, Pia, Brzezinski, Peter
• Format: Article
• Language: English
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1422434112

Cytochrome c oxidase is the terminal electron acceptor in mitochondria and aerobic bacteria, where O 2 reduction is linked to proton pumping across the membrane. Understanding the mechanism by which the enzyme pumps protons requires identification of the so-called proton-loading site, which is the controlling element that assures unidirectionality in the proton flux. The position of this site has been predicted on the basis of theoretical calculations but has not been identified in experimental studies. Here we have used sophisticated biophysical techniques to investigate intraprotein electron and proton transfer in the thermophilic bacterium Thermus thermophilus . The data made it possible to identify the destination of the pumped proton and to unravel the sequence of reactions that leads to proton translocation. The ba 3 -type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O 2 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 ba 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 O 2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c 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 ba 3 cytochrome c oxidase.