Zinc and calcium regulate the mitochondrial redox state

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(Last Updated On: April 4, 2020)
effect of zinc and calcium on mitochondrial redox state
Effect of zinc and calcium on mitochondrial redox state as measured by the real-time flow cytometry. A; control, B; 100µM calcium, C; 100µM zinc and D; combined 100µM zinc and calcium. Image: M.S. Sharaf et al. 2015

Mitochondrial storage of the excess of Zn and Ca ions

Zinc and calcium ions play important roles in maintaining the mitochondrial redox state. Membrane-associated enzymes constituting an electron transport chain are embedded in the inner membrane of the mitochondria. They perform the electron transport during which the NADH donates the electron. Besides that, these enzymes also perform proton translocation from the mitochondrial matrix into the intermembrane space.


Proton translocation leads to the formation of an electrochemical gradient across the inner mitochondrial membrane. ATPase (or complex V) located in the inner mitochondrial membrane transfer proton back to the matrix to maintain the electrochemical balance. ATPase is an enzyme that phosphorylates the ADP into ATP. During the proton transfer back to the matrix, ATPase converts ADP into ATP via oxidative phosphorylation.

Zinc and Calcium ions

Zinc is an essential micronutrient. However, most of the zinc ions found in our system are in the form of metalloproteins. Only a small fraction is available as free ions that contribute to the free zinc ion pool. In the case of excess exposure to the zinc ions, the biological system stores all of the extra zinc ions into the mitochondrial matrix. The excess of zinc ions stored in the matrix may disrupt most of the enzymes responsible for the oxidative phosphorylation, TCA cycle and leads to oxidative stress.

The calcium ion is a key second messenger playing a similar role to that of the zinc. The intracellular ligands bind to most of the calcium ions and some remaining ions are under tight regulation. As in the case of zinc, mitochondria take up all of the excess levels of intracellular calcium ions. Together with the zinc ions, calcium ions stored in the mitochondria impair the normal functions of the mitochondria.

The elevated calcium ions also promote the generation of reactive oxygen species (ROS). And generated ROSs induce the mitochondrial depolarization. The depolarization leads to the opening of the permeability transition pores of the mitochondrial membrane as a result, the proton gradient is dissipated.

More than that, mitochondrial matrix and intermembrane space proteins are also lost from the mitochondria. Cytoplasmic solute particles and water molecules enter into the mitochondria through the permeability transition pores and cause swelling and disruption of the mitochondria.

It has been suggested that zinc enters the mitochondrial matrix through the calcium uniporter, therefore, researchers hypothesized that these two divalent ions interact completely at the mitochondrial level.

To explore the link between zinc and calcium ions in the mitochondrial redox state modulation and functional integrity, researchers carried out an experiment.

Experimental details

Researchers isolated the mitochondria from the liver of the rainbow trout (a type of fish). They measured the protein concentration of the mitochondrial suspension. Researchers also measured the redox state of the mitochondrial using real-time flow cytometry. More than that, they also measured the morphological status using a four-color FACSCalibur flow cytometry equipped with the 488-635 nm lasers.

During the experimentation, the researcher had used H2DCFDA as a probe for the mitochondrial redox state measurement. During the experiment, they used carbonylcyanide-p-trifluomethoxyphenylhydrazone (FCCP) as a mitochondrial protonophore for positive control.

They measured the mitochondrial H2O2 production using an Amplex red assay. During the assay, they added the Amplex red into each of the wells with a final volume of the respiration buffer containing malate and glutamate. They incubate the plate recorded the fluorescence data using the microplate compatible fluorimeter.

The effect of the zinc and calcium on the production of the H2O2 was measured using the antimycin A, an inhibitor of the complex III. Then after, vitamin E and N-acetyl-cysteine were used as antioxidants to access their H2O2 scavenging effects.

Researchers accessed the mitochondrial respiration at 13 °C using the Clark electrode and analyzed the effect of the zinc and calcium on the mitochondrial respiration, using different doses. At last, they measured the mitochondrial size using the transmission electron microscopy. After all these experiments, researchers found that the N-acetyl-cysteine, vitamin E, zinc and calcium all have an anti and pro-oxidant effect based on their doses.


Real-time flow cytometric measurement reveals that the higher dose of the N-acetyl cysteine and vitamin E individually mitigates the oxidation of the mitochondrial redox state. In opposite to that,  their low dose caused a shift of the mitochondrial redox state towards the more oxidized state.

However, the reverse effect was seen in the case of the Zn and the Ca ions. The lower dose of the Zn and Ca alone did not affect the mitochondrial redox state. But in the combination of these ions in a lower dose significantly decreased the oxidation state of the mitochondria. Moreover, the combined high dose of these ions caused strong and time-dependent shifting of the mitochondrial redox state towards the oxidized state.

Effect of zinc and calcium on mitochondrial ultrastructure
Effects of zinc and calcium on mitochondrial ultrastructure as measured by Transmission electron microscopy. A; control, B; 50µM calcium and swelling of mitochondria, C; loss of cristae and vacuolation, (D and E); 50µM zinc with shrinkage and matrix condensation, F; zinc plus calcium 50µM with shrinkage and matrix condensation with widened cristae. Image: M.S. Sharaf et al. 2015

Effect of elevated Zn and Ca ions on morphology

The ions have separate effects on the morphological structure of the mitochondria. Zn alone decreases the size of the mitochondrial. In contrast to that, calcium alone increases mitochondrial size. Increased size of mitochondria causes dilution of the matrix leading to the disruption of the cristae, vacuolation. In addition to that, the diluted matrix leads to the increased separation of the intermembrane space.

Zn and Ca ions individually stimulate the state 3 respiration while at higher dose inhibit the state 3 respiration. But in combination, zinc and calcium at lower doses inhibit the state 3 respiration by 24-89%.

In conclusion, a low dose of the zinc plus calcium has antioxidant properties while at the higher dose they act as a pro-oxidant. Zinc individually or in combination with calcium promotes H2O2 production. However, it doesn’t decouple the mitochondria nor inhibits the complex I. Produced H2O2 are manageable by the natural antioxidants such as glutathione, vitamin E, NADPH, etc.

Reference: Free Radical Biology and Medicine (Zinc and calcium modulate mitochondrial redox state and morphofunctional integrity)

Article DOI: 10.1016/j.freeradbiomed.2015.03.017

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