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The role of external and matrix pH in mitochondrial reactive oxygen species generation.

Selivanov VA, Zeak JA, Roca J, Cascante M, Trucco M, Votyakova TV - J. Biol. Chem. (2008)

Bottom Line: Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin.In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production.The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Associated Unit to Consejo Superior de Investigaciones Científicas, Institute of Biomedicine of the University of Barcelona, Barcelona, Spain.

ABSTRACT
Reactive oxygen species (ROS) generation in mitochondria as a side product of electron and proton transport through the inner membrane is important for normal cell operation as well as development of pathology. Matrix and cytosol alkalization stabilizes semiquinone radical, a potential superoxide producer, and we hypothesized that proton deficiency under the excess of electron donors enhances reactive oxygen species generation. We tested this hypothesis by measuring pH dependence of reactive oxygen species released by mitochondria. The experiments were performed in the media with pH varying from 6 to 8 in the presence of complex II substrate succinate or under more physiological conditions with complex I substrates glutamate and malate. Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin. We found that high pH strongly increased the rate of free radical generation in all of the conditions studied, even when DeltapH=0 in the presence of nigericin. In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production. ROS production increase induced by the alkalization of medium was observed with intact respiring mitochondria as well as in the presence of complex I inhibitor rotenone, which enhanced reactive oxygen species release. The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.

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Scheme complex I to complex III segment of mitochondrial respiratory chain. Redox reactions in electron transport chain and proton translocation are fed by glycolysis and the Krebs cycle, which provide NADH, complex I substrate, and succinate, complex II substrate. Respiratory complex I accepts electrons from NADH, oxidizing it to NAD+, and delivers these electrons to ubiquinone (Q). This delivery is coupled with proton transport from matrix to cytosol. Proton transport in complex III is coupled with electron transport in accordance with the generally accepted ubiquinone/ubiquinol (Q/QH2) cycle mechanism, which is shown in more detail. The overall reaction performed by complex III is as follows, QH2 + 2Cyt Cox + 2H+n ↔ Q + 2Cyt Cred + 4H+p(i.e. it oxidizes ubiquinol, reducing cytochrome c and releasing two H+ to the cytosolic side (positive or p-side; this is reflected in the index of released H+)). In addition, it translocates two protons from matrix (negative or n-side) to cytosol. Ubiquinol (QH2) delivers its first electron to Fe3+, releasing two protons at the p-side of the inner mitochondrial membrane and producing semiquinone radical (SQ-). Then the latter gives its unpaired electron to cytochrome bl, and produced ubiquinone (Q) dissociates from the complex. Free Q binds at the n-side and receives two electrons from cytochrome bh, resulting from oxidation of two QH2 molecules, thus producing subsequently SQ- and QH2, taking protons from the n-side. Dissociation of the produced QH2 accomplishes a round of the cycle.
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fig10: Scheme complex I to complex III segment of mitochondrial respiratory chain. Redox reactions in electron transport chain and proton translocation are fed by glycolysis and the Krebs cycle, which provide NADH, complex I substrate, and succinate, complex II substrate. Respiratory complex I accepts electrons from NADH, oxidizing it to NAD+, and delivers these electrons to ubiquinone (Q). This delivery is coupled with proton transport from matrix to cytosol. Proton transport in complex III is coupled with electron transport in accordance with the generally accepted ubiquinone/ubiquinol (Q/QH2) cycle mechanism, which is shown in more detail. The overall reaction performed by complex III is as follows, QH2 + 2Cyt Cox + 2H+n ↔ Q + 2Cyt Cred + 4H+p(i.e. it oxidizes ubiquinol, reducing cytochrome c and releasing two H+ to the cytosolic side (positive or p-side; this is reflected in the index of released H+)). In addition, it translocates two protons from matrix (negative or n-side) to cytosol. Ubiquinol (QH2) delivers its first electron to Fe3+, releasing two protons at the p-side of the inner mitochondrial membrane and producing semiquinone radical (SQ-). Then the latter gives its unpaired electron to cytochrome bl, and produced ubiquinone (Q) dissociates from the complex. Free Q binds at the n-side and receives two electrons from cytochrome bh, resulting from oxidation of two QH2 molecules, thus producing subsequently SQ- and QH2, taking protons from the n-side. Dissociation of the produced QH2 accomplishes a round of the cycle.

Mentions: Higher matrix pH in the absence of Pi and nigericin (condition 1) at medium pH more than 7 resulted in spontaneous depolarization, probably due to permeability transition as Figs. 3B (squares) and 4A illustrate. In these conditions, mitochondria experience dramatic perturbations with the loss of all gradients across the inner membrane. This resulted in a substantial decrease of ROS release, which requires functional integrity of the mitochondrial membrane (Figs. 3A (squares) and 4B). It can be seen in Fig. 10 that SQ- is not produced if the electron efflux from the FeS center is blocked. On the other hand, matrix pH was shown to be an essential factor in induction of permeability transition (38). Evidence from the literature indicates that the induction of permeability transition may occur in the presence of inorganic phosphate and/or Ca2+ (39). Since these compounds were not present in the medium of condition 1, our results demonstrate that pH itself could regulate permeability transition; this proves the conclusion of our previous theoretical study (40).


The role of external and matrix pH in mitochondrial reactive oxygen species generation.

Selivanov VA, Zeak JA, Roca J, Cascante M, Trucco M, Votyakova TV - J. Biol. Chem. (2008)

Scheme complex I to complex III segment of mitochondrial respiratory chain. Redox reactions in electron transport chain and proton translocation are fed by glycolysis and the Krebs cycle, which provide NADH, complex I substrate, and succinate, complex II substrate. Respiratory complex I accepts electrons from NADH, oxidizing it to NAD+, and delivers these electrons to ubiquinone (Q). This delivery is coupled with proton transport from matrix to cytosol. Proton transport in complex III is coupled with electron transport in accordance with the generally accepted ubiquinone/ubiquinol (Q/QH2) cycle mechanism, which is shown in more detail. The overall reaction performed by complex III is as follows, QH2 + 2Cyt Cox + 2H+n ↔ Q + 2Cyt Cred + 4H+p(i.e. it oxidizes ubiquinol, reducing cytochrome c and releasing two H+ to the cytosolic side (positive or p-side; this is reflected in the index of released H+)). In addition, it translocates two protons from matrix (negative or n-side) to cytosol. Ubiquinol (QH2) delivers its first electron to Fe3+, releasing two protons at the p-side of the inner mitochondrial membrane and producing semiquinone radical (SQ-). Then the latter gives its unpaired electron to cytochrome bl, and produced ubiquinone (Q) dissociates from the complex. Free Q binds at the n-side and receives two electrons from cytochrome bh, resulting from oxidation of two QH2 molecules, thus producing subsequently SQ- and QH2, taking protons from the n-side. Dissociation of the produced QH2 accomplishes a round of the cycle.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2570889&req=5

fig10: Scheme complex I to complex III segment of mitochondrial respiratory chain. Redox reactions in electron transport chain and proton translocation are fed by glycolysis and the Krebs cycle, which provide NADH, complex I substrate, and succinate, complex II substrate. Respiratory complex I accepts electrons from NADH, oxidizing it to NAD+, and delivers these electrons to ubiquinone (Q). This delivery is coupled with proton transport from matrix to cytosol. Proton transport in complex III is coupled with electron transport in accordance with the generally accepted ubiquinone/ubiquinol (Q/QH2) cycle mechanism, which is shown in more detail. The overall reaction performed by complex III is as follows, QH2 + 2Cyt Cox + 2H+n ↔ Q + 2Cyt Cred + 4H+p(i.e. it oxidizes ubiquinol, reducing cytochrome c and releasing two H+ to the cytosolic side (positive or p-side; this is reflected in the index of released H+)). In addition, it translocates two protons from matrix (negative or n-side) to cytosol. Ubiquinol (QH2) delivers its first electron to Fe3+, releasing two protons at the p-side of the inner mitochondrial membrane and producing semiquinone radical (SQ-). Then the latter gives its unpaired electron to cytochrome bl, and produced ubiquinone (Q) dissociates from the complex. Free Q binds at the n-side and receives two electrons from cytochrome bh, resulting from oxidation of two QH2 molecules, thus producing subsequently SQ- and QH2, taking protons from the n-side. Dissociation of the produced QH2 accomplishes a round of the cycle.
Mentions: Higher matrix pH in the absence of Pi and nigericin (condition 1) at medium pH more than 7 resulted in spontaneous depolarization, probably due to permeability transition as Figs. 3B (squares) and 4A illustrate. In these conditions, mitochondria experience dramatic perturbations with the loss of all gradients across the inner membrane. This resulted in a substantial decrease of ROS release, which requires functional integrity of the mitochondrial membrane (Figs. 3A (squares) and 4B). It can be seen in Fig. 10 that SQ- is not produced if the electron efflux from the FeS center is blocked. On the other hand, matrix pH was shown to be an essential factor in induction of permeability transition (38). Evidence from the literature indicates that the induction of permeability transition may occur in the presence of inorganic phosphate and/or Ca2+ (39). Since these compounds were not present in the medium of condition 1, our results demonstrate that pH itself could regulate permeability transition; this proves the conclusion of our previous theoretical study (40).

Bottom Line: Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin.In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production.The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Associated Unit to Consejo Superior de Investigaciones Científicas, Institute of Biomedicine of the University of Barcelona, Barcelona, Spain.

ABSTRACT
Reactive oxygen species (ROS) generation in mitochondria as a side product of electron and proton transport through the inner membrane is important for normal cell operation as well as development of pathology. Matrix and cytosol alkalization stabilizes semiquinone radical, a potential superoxide producer, and we hypothesized that proton deficiency under the excess of electron donors enhances reactive oxygen species generation. We tested this hypothesis by measuring pH dependence of reactive oxygen species released by mitochondria. The experiments were performed in the media with pH varying from 6 to 8 in the presence of complex II substrate succinate or under more physiological conditions with complex I substrates glutamate and malate. Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin. We found that high pH strongly increased the rate of free radical generation in all of the conditions studied, even when DeltapH=0 in the presence of nigericin. In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production. ROS production increase induced by the alkalization of medium was observed with intact respiring mitochondria as well as in the presence of complex I inhibitor rotenone, which enhanced reactive oxygen species release. The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.

Show MeSH
Related in: MedlinePlus