Abstract
This study addresses the eventual consequence of cytochrome c oxidase (CytOx) inhibition by ATP at high ATP/ADP ratio in isolated rat heart mitochondria. Earlier, it has been demonstrated that the mechanism of allosteric ATP inhibition of CytOx is one of the key regulations of mitochondrial functions. It is relevant that aiming to maintain a high ATP/ADP ratio for the measurement of CytOx activity effectuating the enzymatic inhibition as well as mitochondrial respiration, optimal concentration of mitochondria is critically important. Likewise, only at this concentration, were the differences in ΔΨm and ROS concentrations measured under various conditions significant. Moreover, when CytOx activity was inhibited in the presence of ATP, mitochondrial respiration and ΔΨm both remained static, while the ROS production was markedly decreased. Consubstantial results were found when the electron transport chain was inhibited by antimycin A, letting only CytOx remain functional to support the energy production. This seems to corroborate that the decrease in mitochondrial ROS production is solely the effect of ATP binding to CytOx which results in static respiration as well as membrane potential.
Highlights
Life requires energy, which is mostly supplied by ATP
In order to exclude the fact if it is due to the ATP/ADP ratio dependent on the mitochondrial protein concentration, a 20 times lower concentration of mitochondria was used to measure the respiration (Figure 1D), ∆Ψm (Figure 1E) and reactive oxygen species (ROS)
The ROS concentrations remained the same in the absence and presence of cytochrome c when ATP + regenerating system (RS) was added. These results indicate that ROS production decreases tremendously just by adding ATP + RS independent of mitochondrial concentrations as well as the presence/absence of cytochrome c
Summary
Life requires energy, which is mostly supplied by ATP. Oxidative phosphorylation has to be highly regulated in eukaryotic cells because the requirement of ATP utilization may change by a factor of 5–10 depending on the energy demand [1,2]. The cell faces ultimate deleterious effects of excessive respiration in the form of an increased accompanied production of reactive oxygen species (ROS), resulting in oxidative stress. The role of cytochrome c oxidase in the regulation of respiration and membrane potential via excessive ATP production has already been proposed and described [7] apart from binding of different ligands and their roles in switching between active and relaxed state of the enzyme along with molecular conformational change [8]. It is relevant that a regulatory missing link was found in the form of a mechanism where
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