Mutation of a single residue in the ba3 oxidase specifically impairs protonation of the pump site

Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3397-402. doi: 10.1073/pnas.1422434112. Epub 2015 Mar 2.

Abstract

The ba3-type cytochrome c oxidase from Thermus thermophilus 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 ba3 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 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 ba3 cytochrome c oxidase.

Keywords: cytochrome aa3; cytochrome c oxidase; electron transfer; membrane protein; respiration.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspartic Acid / genetics*
  • Cytochrome b Group / chemistry
  • Cytochrome b Group / genetics*
  • Electron Transport Complex IV / chemistry
  • Electron Transport Complex IV / genetics*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Mutation / genetics*
  • Proton Pumps / genetics*
  • Protons*
  • Thermus thermophilus / enzymology*
  • Time Factors

Substances

  • Cytochrome b Group
  • Proton Pumps
  • Protons
  • Aspartic Acid
  • cytochrome ba3
  • Electron Transport Complex IV