Cytochrome c Oxidase Inhibition by ATP Decreases Mitochondrial ROS Production

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 ΔΨ_m and ROS concentrations measured under various conditions significant. Moreover, when CytOx activity was inhibited in the presence of ATP, mitochondrial respiration and ΔΨ_m 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.

Keywords: ATP; ROS; cytochrome c oxidase; mitochondrial membrane potential.

Figure 1

Correlation of mitochondrial respiration, membrane potential and ROS production. Mitochondrial oxygen consumption (respiration) rates were measured by polarography in the presence of endogenous fuel substrates using two different concentrations of mitochondria, i.e., 1.128 mg/mL ± 0.07 and 0.054 mg/mL ± 0.004 ((A,D), respectively) and were correlated to the corresponding spectrofluorometric measurements of membrane potential ((B,E), respectively) as well as to the ROS productions (C,F). The required mitochondrial concentration (n = 4 individual experiments) was added into the total volume of 0.5 mL of kinetics measuring buffer with or without substrate additions as indicated. Finally, samples were measured in duplicates after mixing with the corresponding fluorescent dyes. In addition, mitochondrial membrane potential (G) and ROS measurements (H) were also performed by flow cytometry using 0.054 mg/mL ± 0.004 (n = 5 individual experiments), and were compared to the corresponding polarographic measurements of mitochondrial respiration (D). A standard one-way ANOVA with Tukey’s Post hoc analysis was applied to the data. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. The concentrations of added substrates were as follows; 5 mM ADP, 5 mM ATP + RS (10 mM phosphoenolpyruvate + 160 U/mL pyruvate kinase). Rat heart mitochondria (RHM) without addition of substrates was used as a control (Ctrl).

Figure 2

Variation in CytOx kinetics depending on mitochondrial protein concentrations. The activity of CytOx kinetics was measured using 1.128, 0.562, 0.276 and 0.054 mg/mL mitochondrial concentrations at 0, 5, 10 and 40 µM cytochrome c (substrate of CytOx) in the presence of 18 mM ascorbate, which was used to reduce cytochrome c. (A) in the presence of 5 mM ADP, (B) in the presence of 5 mM ATP + RS (10 mM PEP + 160 U/mL PK). The activity of CytOx measured in the presence of ADP as well as ATP + RS at lower (0.054 mg/mL) concentration of RHM was significantly different (p ≤ 0.0001) from all other higher mitochondrial protein concentrations when a regular two-way ANOVA with Bonferroni Post hoc test was applied to the data consisting of n = 4 individual experiments.

Figure 3

Comparison of CytOx kinetics to mitochondrial membrane potential and ROS production measurements. The kinetics of CytOx activity was measured in 1.128 mg/mL ± 0.07 mitochondria (A) and was correlated with the membrane potential (B) and ROS productions (C) performed by spectrofluorometry (n = 4 individual experiments). Similarly, measurements were also performed using 0.054 mg/mL ± 0.004 mitochondria, i.e., CytOx kinetics (D), ΔΨ_m (E) and ROS (F). Additionally, membrane potential (G) and ROS production (H) were also measured by flow cytometry (n = 3 individual experiments) in 0.054 mg/mL ± 0.004 mitochondria. The concentrations of added substrates were: 5 mM ADP or 5 mM ATP + RS (10 mM PEP + 160 U/mL PK). Each sample was measured at 0, 5, 10 or 40 µM cytochrome c used as a substrate for CytOx. All measurements were performed in the presence of 18 mM ascorbate which was added to reduce cytochrome c. A regular two-way ANOVA with Bonferroni Post hoc test was applied and following p values were found showing significant differences between different groups based on cytochrome c concentrations; p ≤ 0.0001 for (A); p ≤ 0.0001 for (B); p ≤ 0.0001 for (C); p ≤ 0.01 for (D); p ≤ 0.05 for (E) and p ≤ 0.0001 for (F). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 4

Effect of antimycin A on CytOx kinetics, mitochondrial membrane potential and ROS production. Samples were prepared by adding 0.054 mg/mL ± 0.004 freshly isolated rat heart mitochondria to a total volume of 0.5 mL kinetics measuring buffer in the presence or absence of 45 µM antimycin A. (A) The kinetics of CytOx activity was measured polarographically in the absence of nucleotides (control) or in the presence of 5 mM ADP or 5 mM ATP + RS (10 mM PEP + 160 U/mL PK) at increasing cytochrome c concentrations of 0, 5, 10 and 40 µM using 18 mM ascorbate to reduce cytochrome c. (B) The CytOx kinetics was measured in the presence of 45 µM antimycin A. For n = 5 individual experiments, a regular two-way ANOVA with Bonferroni Post hoc test was applied. Significant differences (p ≤ 0.0001) were found (in case of both A and B) for ATP + RS in comparison to control and ADP, while the differences between the control and ADP were not significant. Furthermore, cytochrome c concentrations were found to be significantly (p ≤ 0.0001) affecting the oxygen consumption rates. Mitochondrial membrane potential (C) and ROS production (D) were performed by flow cytometry. A standard one-way ANOVA with Tukey’s Post hoc analysis was performed with data from n = 5 measurements (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Figure 5

Schematic representation of variations in mitochondrial functional components under different physiological states, e.g., relaxed, activated and activated under work load.