Limits...
Single Enzyme Experiments Reveal a Long-Lifetime Proton Leak State in a Heme-Copper Oxidase.

Li M, Jørgensen SK, McMillan DG, Krzemiński Ł, Daskalakis NN, Partanen RH, Tutkus M, Tuma R, Stamou D, Hatzakis NS, Jeuken LJ - J. Am. Chem. Soc. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, University of Leeds , LS2 9JT Leeds, U.K.

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.

No MeSH data available.


Related in: MedlinePlus

Distribution of proton translocation ratesfor (top) wild-typecytochrome bo3 (middle) E286C cytochrome bo3 and (bottom) wild-type cytochrome bo3 in the presence of valinomycin. Cytochrome bo3 was orientated both ways in the single-enzymeproteoliposomes and relative frequencies are separated for “protonuptake” (positive rates) and “proton release”(negative rates). The rates are determined from the slope of the analyzeddata for which examples are shown in Figure 2. The error bars represent the standard errorof the mean.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4697922&req=5

fig3: Distribution of proton translocation ratesfor (top) wild-typecytochrome bo3 (middle) E286C cytochrome bo3 and (bottom) wild-type cytochrome bo3 in the presence of valinomycin. Cytochrome bo3 was orientated both ways in the single-enzymeproteoliposomes and relative frequencies are separated for “protonuptake” (positive rates) and “proton release”(negative rates). The rates are determined from the slope of the analyzeddata for which examples are shown in Figure 2. The error bars represent the standard errorof the mean.

Mentions: The pH values were observed to both increaseand decrease (Figure 2a and 2b, respectively; hereafter these proteoliposomeswill be describedas “active” liposomes or vesicles), indicating cytochrome bo3 was present in both orientations in the proteoliposomes.About 76% of active vesicles show a pH increase consistent with a“right-side out” orientation. The single-enzyme tracesreveal a distribution in turnover rates (Figure 3), while in more than 10% of cases, cytochrome bo3 was observed to abruptly halt proton uptake/release(Figure 2e). Such dynamicheterogeneity and “stalling” of activity was observedfor enzymes in both orientations and is a feature typically observedfor (single) enzymes.29 Importantly, however,a significant fraction of our single enzyme traces (7.2 ± 1.5%)also show abrupt turns in pH changes, rapidly destroying formed ΔpHeven when the potential was still applied to reduce the ubiquinonepool. Such events were observed for cytochrome bo3 in both orientations, as shown in Figure 2c and d.


Single Enzyme Experiments Reveal a Long-Lifetime Proton Leak State in a Heme-Copper Oxidase.

Li M, Jørgensen SK, McMillan DG, Krzemiński Ł, Daskalakis NN, Partanen RH, Tutkus M, Tuma R, Stamou D, Hatzakis NS, Jeuken LJ - J. Am. Chem. Soc. (2015)

Distribution of proton translocation ratesfor (top) wild-typecytochrome bo3 (middle) E286C cytochrome bo3 and (bottom) wild-type cytochrome bo3 in the presence of valinomycin. Cytochrome bo3 was orientated both ways in the single-enzymeproteoliposomes and relative frequencies are separated for “protonuptake” (positive rates) and “proton release”(negative rates). The rates are determined from the slope of the analyzeddata for which examples are shown in Figure 2. The error bars represent the standard errorof the mean.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697922&req=5

fig3: Distribution of proton translocation ratesfor (top) wild-typecytochrome bo3 (middle) E286C cytochrome bo3 and (bottom) wild-type cytochrome bo3 in the presence of valinomycin. Cytochrome bo3 was orientated both ways in the single-enzymeproteoliposomes and relative frequencies are separated for “protonuptake” (positive rates) and “proton release”(negative rates). The rates are determined from the slope of the analyzeddata for which examples are shown in Figure 2. The error bars represent the standard errorof the mean.
Mentions: The pH values were observed to both increaseand decrease (Figure 2a and 2b, respectively; hereafter these proteoliposomeswill be describedas “active” liposomes or vesicles), indicating cytochrome bo3 was present in both orientations in the proteoliposomes.About 76% of active vesicles show a pH increase consistent with a“right-side out” orientation. The single-enzyme tracesreveal a distribution in turnover rates (Figure 3), while in more than 10% of cases, cytochrome bo3 was observed to abruptly halt proton uptake/release(Figure 2e). Such dynamicheterogeneity and “stalling” of activity was observedfor enzymes in both orientations and is a feature typically observedfor (single) enzymes.29 Importantly, however,a significant fraction of our single enzyme traces (7.2 ± 1.5%)also show abrupt turns in pH changes, rapidly destroying formed ΔpHeven when the potential was still applied to reduce the ubiquinonepool. Such events were observed for cytochrome bo3 in both orientations, as shown in Figure 2c and d.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, University of Leeds , LS2 9JT Leeds, U.K.

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.

No MeSH data available.


Related in: MedlinePlus