Electronation-dependent structural change at the proton exit side of cytochrome c oxidase as revealed by site-directed fluorescence labeling.

Cytochrome c oxidase (CcO), 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, CcO 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 CcO through the development of a new homologous mutagenesis/expression system in Paracoccus denitrificans, which allows mutagenesis of CcO subunits 1, 2, and 3. Our system provides true single thiol-reactive CcO 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 CcO for cysteines. We show redox-dependent wetting changes at the proton uptake channel and increased polarity at the proton exit side of CcO upon electronation. We suggest an electronation-dependent conformational change to play a role in proton exit from CcO.