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Electronation‐dependent structural change at the proton exit side of cytochrome c oxidase as revealed by site‐directed fluorescence labeling
Author(s) -
Wolf Alexander,
Wonneberg Juliane,
Balke Jens,
Alexiev Ulrike
Publication year - 2020
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.15084
Subject(s) - paracoccus denitrificans , chemistry , cytochrome c oxidase , protonation , electron transport complex iv , mutagenesis , stereochemistry , protein subunit , respiratory chain , directed mutagenesis , biophysics , biochemistry , enzyme , mutant , biology , ion , organic chemistry , gene
Cytochrome c oxidase (C c O), the terminal enzyme of the respiratory chain of mitochondria and many aerobic prokaryotes that function as a redox‐coupled proton pump, catalyzes the reduction of molecular oxygen to water. As part of the respiratory chain, C c O contributes to the proton motive force driving ATP synthesis. While many aspects of the enzyme’s catalytic mechanisms have been established, a clear picture of the proton exit pathway(s) remains elusive. Here, we aim to gain insight into the molecular mechanisms of C c O through the development of a new homologous mutagenesis/expression system in Paracoccus denitrificans , which allows mutagenesis of C c O subunits 1, 2, and 3. Our system provides true single thiol‐reactive C c O variants in a three‐subunit base variant with unique labeling sites for the covalent attachment of reporter groups sensitive to nanoenvironmental factors like protonation, polarity, and hydration. To this end, we exchanged six residues on both membrane sides of C c O for cysteines. We show redox‐dependent wetting changes at the proton uptake channel and increased polarity at the proton exit side of C c O upon electronation. We suggest an electronation‐dependent conformational change to play a role in proton exit from C c O.