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Electrochemical detection of intracellular and cell membrane redox systems in Saccharomyces cerevisiae.

Rawson FJ, Downard AJ, Baronian KH - Sci Rep (2014)

Bottom Line: After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells.Four of the mediators inhibit electron transfer from S. cerevisiae.Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Biophysics and Surfaces Analysis, School of Pharmacy, University of Nottingham, University Park, Nottingham B15 2TT UK [2] Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.

ABSTRACT
Redox mediators can interact with eukaryote cells at a number of different cell locations. While cell membrane redox centres are easily accessible, the redox centres of catabolism are situated within the cytoplasm and mitochondria and can be difficult to access. We have systematically investigated the interaction of thirteen commonly used lipophilic and hydrophilic mediators with the yeast Saccharomyces cerevisiae. A double mediator system is used in which ferricyanide is the final electron acceptor (the reporter mediator). After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells. The plateau current at 425 mV vs Ag/AgCl gives the analytical signal. The results show that five of the mediators interact with at least three different trans Plasma Membrane Electron Transport systems (tPMETs), and that four mediators cross the plasma membrane to interact with cytoplasmic and mitochondrial redox molecules. Four of the mediators inhibit electron transfer from S. cerevisiae. Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

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Related in: MedlinePlus

Plot of mean steady state currents (iss) obtained from linear sweep voltammograms at 425 mV vs Ag/AgCl versus concentration partition coefficients (log P) for BQ, SFN, GC, MD, DCIP, MR, and N,N,N′,N′-TMPD.
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f4: Plot of mean steady state currents (iss) obtained from linear sweep voltammograms at 425 mV vs Ag/AgCl versus concentration partition coefficients (log P) for BQ, SFN, GC, MD, DCIP, MR, and N,N,N′,N′-TMPD.

Mentions: The conversion of [Fe(CN)6]3− to [Fe(CN)6]4− in the presence of 1-M-PMS and resorufin is not significantly different from the [Fe(CN)6]3− cellular control. Tan et al.6 suggest that 1-M-PMS interacts with the same tPMET sites as [Fe(CN)6]3−. Our result supports this proposal because, if 1-M-PMS (and resorufin) competes with [Fe(CN)6]3− for the same tPMET electrons, [Fe(CN)6]3− will oxidise any reduced 1-M-PMS or resorufin resulting in a signal that is of the same magnitude as the [Fe(CN)6]3− control signal. Figure 4A shows that there is not a strong correlation between signal size and log P. For example log P of MD is less than that of methyl red, yet the MD current is almost 9 times larger than the methyl red current. Considering the benzoquinone, safranine, gallocyanine and methyl red group, methyl red is an azo dye and Ramalho et al. have shown that the ferri-reductase tPMET system of S. cerevisiae is involved in the extracellular reduction of azo dyes. The study found that deletion of the FRE 1 gene encoding the tPMET protein does not completely remove azo activity and hence the reduction is probably not totally dependent on tPMETs21. Jahdav et al.22 also report that reduction of methyl red remains extracellular in S. cerevisiae. There is one report on the use of safranine as a tool for investigating variations in membrane potential by fluorescence microscopy however in that study, the cells were first permeabilised with digitonin23. We have not found relevant information on the redox interaction of benzoquinone and gallocyanine with eukaryote cells. Nevertheless, the currents for these mediators are significantly greater than for [Fe(CN)6]3− alone, indicating that they probably interact with tPMET sites not accessible to [Fe(CN)6]3−, 1-M-PMS and resofurin. For methyl red, benzoquinone, gallocyanine and safranine, the anodic current increases as the formal potential increases (Table 1). Figure 5A shows a plot of the log of the mean steady-state current versus the potential of these (and other) mediators. A linear relationship between these parameters is expected if the rate of reduction of the secondary mediator is controlled by the difference in potential between the formal potentials of the mediator and the cellular redox site(s), that is, the thermodynamic driving force. A R2 value of 0.895 is obtained for a linear fit to the data for methyl red, benzoquinone, gallocyanine and safranine, however with the removal of the benzoquinone datum point, the R2 value becomes 0.985 (Figure 5A, inset). This analysis is consistent with interaction of gallocyanine, methyl red and safranine with the same tPMET site or with different tPMET sites with the same redox potential, whereas benzoquinone interacts with a tPMET site that has a different redox potential. Considering the data in a different way and assuming that electron transfer from cellular redox sites to the secondary mediator is fast and not rate-limited, the mean current should depend on the number of tPMET sites, which are thermodynamically able to reduce the mediator. Figure 5B shows a plot of mean current generated versus formal potential of the mediators. This plot suggests that methyl red, benzoquinone, gallocyanine and safranine may interact with additional tPMET sites to that accessible to FeIII, 1-M-PMS and resofurin. Methyl red accepts electrons from three additional tPMET sites, benzoquinone and gallocyanine from two and safranine from one. Both of the analyses outlined above support the notion that these mediators are interacting externally with at least two tPMET sites additional to that accessible to FeIII, 1-M-PMS and resofurin (Figure 1, pathway 4). We tentatively propose that the resofurin, PMS and [Fe(CN)6]3− signals are not related to their redox potentials because these mediators may be interacting with non-voltage gated tPMETs, whereas the other mediators are interacting with voltage gated tPMETs.


Electrochemical detection of intracellular and cell membrane redox systems in Saccharomyces cerevisiae.

Rawson FJ, Downard AJ, Baronian KH - Sci Rep (2014)

Plot of mean steady state currents (iss) obtained from linear sweep voltammograms at 425 mV vs Ag/AgCl versus concentration partition coefficients (log P) for BQ, SFN, GC, MD, DCIP, MR, and N,N,N′,N′-TMPD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048887&req=5

f4: Plot of mean steady state currents (iss) obtained from linear sweep voltammograms at 425 mV vs Ag/AgCl versus concentration partition coefficients (log P) for BQ, SFN, GC, MD, DCIP, MR, and N,N,N′,N′-TMPD.
Mentions: The conversion of [Fe(CN)6]3− to [Fe(CN)6]4− in the presence of 1-M-PMS and resorufin is not significantly different from the [Fe(CN)6]3− cellular control. Tan et al.6 suggest that 1-M-PMS interacts with the same tPMET sites as [Fe(CN)6]3−. Our result supports this proposal because, if 1-M-PMS (and resorufin) competes with [Fe(CN)6]3− for the same tPMET electrons, [Fe(CN)6]3− will oxidise any reduced 1-M-PMS or resorufin resulting in a signal that is of the same magnitude as the [Fe(CN)6]3− control signal. Figure 4A shows that there is not a strong correlation between signal size and log P. For example log P of MD is less than that of methyl red, yet the MD current is almost 9 times larger than the methyl red current. Considering the benzoquinone, safranine, gallocyanine and methyl red group, methyl red is an azo dye and Ramalho et al. have shown that the ferri-reductase tPMET system of S. cerevisiae is involved in the extracellular reduction of azo dyes. The study found that deletion of the FRE 1 gene encoding the tPMET protein does not completely remove azo activity and hence the reduction is probably not totally dependent on tPMETs21. Jahdav et al.22 also report that reduction of methyl red remains extracellular in S. cerevisiae. There is one report on the use of safranine as a tool for investigating variations in membrane potential by fluorescence microscopy however in that study, the cells were first permeabilised with digitonin23. We have not found relevant information on the redox interaction of benzoquinone and gallocyanine with eukaryote cells. Nevertheless, the currents for these mediators are significantly greater than for [Fe(CN)6]3− alone, indicating that they probably interact with tPMET sites not accessible to [Fe(CN)6]3−, 1-M-PMS and resofurin. For methyl red, benzoquinone, gallocyanine and safranine, the anodic current increases as the formal potential increases (Table 1). Figure 5A shows a plot of the log of the mean steady-state current versus the potential of these (and other) mediators. A linear relationship between these parameters is expected if the rate of reduction of the secondary mediator is controlled by the difference in potential between the formal potentials of the mediator and the cellular redox site(s), that is, the thermodynamic driving force. A R2 value of 0.895 is obtained for a linear fit to the data for methyl red, benzoquinone, gallocyanine and safranine, however with the removal of the benzoquinone datum point, the R2 value becomes 0.985 (Figure 5A, inset). This analysis is consistent with interaction of gallocyanine, methyl red and safranine with the same tPMET site or with different tPMET sites with the same redox potential, whereas benzoquinone interacts with a tPMET site that has a different redox potential. Considering the data in a different way and assuming that electron transfer from cellular redox sites to the secondary mediator is fast and not rate-limited, the mean current should depend on the number of tPMET sites, which are thermodynamically able to reduce the mediator. Figure 5B shows a plot of mean current generated versus formal potential of the mediators. This plot suggests that methyl red, benzoquinone, gallocyanine and safranine may interact with additional tPMET sites to that accessible to FeIII, 1-M-PMS and resofurin. Methyl red accepts electrons from three additional tPMET sites, benzoquinone and gallocyanine from two and safranine from one. Both of the analyses outlined above support the notion that these mediators are interacting externally with at least two tPMET sites additional to that accessible to FeIII, 1-M-PMS and resofurin (Figure 1, pathway 4). We tentatively propose that the resofurin, PMS and [Fe(CN)6]3− signals are not related to their redox potentials because these mediators may be interacting with non-voltage gated tPMETs, whereas the other mediators are interacting with voltage gated tPMETs.

Bottom Line: After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells.Four of the mediators inhibit electron transfer from S. cerevisiae.Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Biophysics and Surfaces Analysis, School of Pharmacy, University of Nottingham, University Park, Nottingham B15 2TT UK [2] Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.

ABSTRACT
Redox mediators can interact with eukaryote cells at a number of different cell locations. While cell membrane redox centres are easily accessible, the redox centres of catabolism are situated within the cytoplasm and mitochondria and can be difficult to access. We have systematically investigated the interaction of thirteen commonly used lipophilic and hydrophilic mediators with the yeast Saccharomyces cerevisiae. A double mediator system is used in which ferricyanide is the final electron acceptor (the reporter mediator). After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells. The plateau current at 425 mV vs Ag/AgCl gives the analytical signal. The results show that five of the mediators interact with at least three different trans Plasma Membrane Electron Transport systems (tPMETs), and that four mediators cross the plasma membrane to interact with cytoplasmic and mitochondrial redox molecules. Four of the mediators inhibit electron transfer from S. cerevisiae. Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.

Show MeSH
Related in: MedlinePlus