Abstract
Heme-copper oxidases (HCOs) are key enzymes in prokaryotes and eukaryotes for energy production during aerobic respiration. They catalyze the reduction of the terminal electron acceptor, oxygen, and utilize the Gibbs free energy to transport protons across a membrane to generate a proton (ΔpH) and electrochemical gradient termed proton motive force (PMF), which provides the driving force for the adenosine triphosphate (ATP) synthesis. Excessive PMF is known to limit the turnover of HCOs, but the molecular mechanism of this regulatory feedback remains relatively unexplored. Here we present a single-enzyme study that reveals that cytochrome bo3 from Escherichia coli, an HCO closely homologous to Complex IV in human mitochondria, can enter a rare, long-lifetime leak state during which proton flow is reversed. The probability of entering the leak state is increased at higher ΔpH. By rapidly dissipating the PMF, we propose that this leak state may enable cytochrome bo3, and possibly other HCOs, to maintain a suitable ΔpH under extreme redox conditions.
Highlights
One of the main roles of oxidative phosphorylation in mitochondria and bacteria is maintaining a proton-motive force (PMF) across the inner or plasma membrane, thereby providing most of the energy for the synthesis of adenosine triphosphate (ATP) in aerobic environments
Type-A heme-copper oxygen reductases (HCOs) possess two conserved proton channels, known as the K- and D-channel, both leading to the binuclear center (BNC).[2−5] The K-channel facilitates the transportation of “chemical protons” that react with dioxygen to form water; the D-channel facilitates both the uptake of “chemical protons” and the transportation of all the pumped protons.[6−9] Both channels take up protons from the same side of the membrane: the matrix side in mitochondria or the cytoplasmic side in bacteria.[2,3]
The gold is modified with a self-assembled monolayer of 6-mercapto-hexanol, which renders it hydrophilic and allows immobilization of the proteoliposomes, that retain the pH sensitive fluorophore
Summary
One of the main roles of oxidative phosphorylation in mitochondria and bacteria is maintaining a proton-motive force (PMF) across the inner or plasma membrane, thereby providing most of the energy for the synthesis of adenosine triphosphate (ATP) in aerobic environments. Complex IV and a bacterial homologue, cytochrome bo[3] from Escherichia coli, belong to the A1 subgroup of Type-A HCOs, which typically pump one proton per electron, four protons per dioxygen molecule.[1] The reduction of dioxygen is further coupled to the uptake/release of four charges (protons and/or electrons), adding up to a total of eight charges transported across the membrane for each dioxygen that is reduced. Type-A HCOs possess two conserved proton channels, known as the K- and D-channel, both leading to the BNC.[2−5] The K-channel facilitates the transportation of “chemical protons” that react with dioxygen to form water; the D-channel facilitates both the uptake of “chemical protons” and the transportation of all the pumped protons.[6−9] Both channels take up protons from the same side of the membrane: the matrix side in mitochondria or the cytoplasmic side in bacteria.[2,3]
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