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

The relative frequency (in percentage) of enteringthe leak stateduring turnover in relation to the leaking rate and the ΔpHformed prior to the start of the leaking events. These figures aretwo-dimensional histograms generated in the same way as the conventionalone-dimensional histogram (the color of the pixels is equivalent tothe height of the bars in histogram bar chart). The sum of the relativefrequency values in each heat map equals to the total relative frequencyof proteoliposomes entering the leak state in the corresponding experiment.The ΔpH is the intravesicular pH minus the pH of the bulk solution(set constant at 7.4).
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fig5: The relative frequency (in percentage) of enteringthe leak stateduring turnover in relation to the leaking rate and the ΔpHformed prior to the start of the leaking events. These figures aretwo-dimensional histograms generated in the same way as the conventionalone-dimensional histogram (the color of the pixels is equivalent tothe height of the bars in histogram bar chart). The sum of the relativefrequency values in each heat map equals to the total relative frequencyof proteoliposomes entering the leak state in the corresponding experiment.The ΔpH is the intravesicular pH minus the pH of the bulk solution(set constant at 7.4).

Mentions: The single-enzyme experiments show that the relative frequencyby which E286C enters the leak state is significantly reduced to 0.6%(0.5% and 1% in two independent data sets each containing eight and20 movies, respectively), compared to 7.2% for wild-type cytochrome bo3. Besides the significantly reduced percentageof leak events observed for E286C cytochrome bo3, the leaking rates are also significantly lower for E286C(25.5 ± 4.1 H+/s vs 102.3 ± 11.2 H+/s for the wild-type, Figure 5). We propose that these results further substantiate thatthe leak events observed from wild-type cytochrome bo3 are caused by a leak state of the proton pump and notby oppositely orientated enzymes or pore formation in the lipid membrane.Although the results from the E286C mutant confirm that protons leakthrough the protein, they do not unambiguously show that protons flowvia the D-pathway and more work is needed to identify the leak pathway.Because this mutant is less active than WT, the E286C mutation couldallosterically reduce the probability that a leak state is formed.Indeed, on average, the lifetime of proton uptake/release in E286Ccytochrome is about 25% longer than WT (Figure S4), but as lifetimes longer than 100s were capped becauseof the technical limitations on for how long an experiment can continue,this should be considered a lower limit. Therefore, there is an indicationthat the E286C mutant may have a longer lifetime for proton uptake/release.Related to this, the “stalling” behavior was observedin only 4% of the active vesicles.


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)

The relative frequency (in percentage) of enteringthe leak stateduring turnover in relation to the leaking rate and the ΔpHformed prior to the start of the leaking events. These figures aretwo-dimensional histograms generated in the same way as the conventionalone-dimensional histogram (the color of the pixels is equivalent tothe height of the bars in histogram bar chart). The sum of the relativefrequency values in each heat map equals to the total relative frequencyof proteoliposomes entering the leak state in the corresponding experiment.The ΔpH is the intravesicular pH minus the pH of the bulk solution(set constant at 7.4).
© Copyright Policy
Related In: Results  -  Collection

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

fig5: The relative frequency (in percentage) of enteringthe leak stateduring turnover in relation to the leaking rate and the ΔpHformed prior to the start of the leaking events. These figures aretwo-dimensional histograms generated in the same way as the conventionalone-dimensional histogram (the color of the pixels is equivalent tothe height of the bars in histogram bar chart). The sum of the relativefrequency values in each heat map equals to the total relative frequencyof proteoliposomes entering the leak state in the corresponding experiment.The ΔpH is the intravesicular pH minus the pH of the bulk solution(set constant at 7.4).
Mentions: The single-enzyme experiments show that the relative frequencyby which E286C enters the leak state is significantly reduced to 0.6%(0.5% and 1% in two independent data sets each containing eight and20 movies, respectively), compared to 7.2% for wild-type cytochrome bo3. Besides the significantly reduced percentageof leak events observed for E286C cytochrome bo3, the leaking rates are also significantly lower for E286C(25.5 ± 4.1 H+/s vs 102.3 ± 11.2 H+/s for the wild-type, Figure 5). We propose that these results further substantiate thatthe leak events observed from wild-type cytochrome bo3 are caused by a leak state of the proton pump and notby oppositely orientated enzymes or pore formation in the lipid membrane.Although the results from the E286C mutant confirm that protons leakthrough the protein, they do not unambiguously show that protons flowvia the D-pathway and more work is needed to identify the leak pathway.Because this mutant is less active than WT, the E286C mutation couldallosterically reduce the probability that a leak state is formed.Indeed, on average, the lifetime of proton uptake/release in E286Ccytochrome is about 25% longer than WT (Figure S4), but as lifetimes longer than 100s were capped becauseof the technical limitations on for how long an experiment can continue,this should be considered a lower limit. Therefore, there is an indicationthat the E286C mutant may have a longer lifetime for proton uptake/release.Related to this, the “stalling” behavior was observedin only 4% of the active vesicles.

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