Structural Elements Involved in Proton Translocation by Cytochrome c Oxidase as Revealed by Backbone Amide Hydrogen-Deuterium Exchange of the E286H Mutant
详细信息 查看全文
- 作者：Laura S. Busenlehner ; Gisela Brä ; ndé ; n ; Ida Namslauer ; Peter Brzezinski ; Richard N. Armstrong
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：January 8, 2008
- 年：2008
- 卷：47
- 期：1
- 页码：73 - 83
- 全文大小：449K
- 年卷期：v.47,no.1(January 8, 2008)
- ISSN：1520-4995
文摘
Cytochrome c oxidase is the terminal electron acceptor in the respiratory chains of aerobicorganisms and energetically couples the reduction of oxygen to water to proton pumping across themembrane. The mechanisms of proton uptake, gating, and pumping have yet to be completely elucidatedat the molecular level for these enzymes. For Rhodobacter sphaeroides CytcO (cytochrome aa3), it appearsas though the E286 side chain of subunit I is a branching point from which protons are shuttled either tothe catalytic site for O2 reduction or to the acceptor site for pumped protons. Amide hydrogen-deuteriumexchange mass spectrometry was used to investigate how mutation of this key branching residue to histidine(E286H) affects the structures and dynamics of four redox intermediate states. A functional characterizationof this mutant reveals that E286H CytcO retains ~1% steady-state activity that is uncoupled from protonpumping and that proton transfer from H286 is significantly slowed. Backbone amide H-D exchangekinetics indicates that specific regions of CytcO, perturbed by the E286H mutation, are likely to be involvedin proton gating and in the exit pathway for pumped protons. The results indicate that redox-dependentconformational changes around E286 are essential for internal proton transfer. E286H CytcO, however,is incapable of these specific conformational changes and therefore is insensitive to the redox state of theenzyme. These data support a model where the side chain conformation of E286 controls protontranslocation in CytcO through its interactions with the proton gate, which directs the flow of protonseither to the active site or to the exit pathway. In the E286H mutant, the proton gate does not functionproperly and the exit channel is unresponsive. These results provide new insight into the structure andmechanism of proton translocation by CytcO.