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Application of N,N,N',N'-Tetramethyl-p-Phenylenediamine and α,ω-Hexadecanedioic Acid for Determination of the H+/O Ratios of Complexes III and IV of the Liver Mitochondrial Respiratory Chain under Free Respiration Conditions

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Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology Aims and scope

Abstract—

Stimulation of mitochondrial respiration in state 4 without changes in passive proton leakage is known to be accompanied by a decrease in the H+/O ratio. In the present work, it was found that during the oxidation of succinate by liver mitochondria, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and α,ω-hexadecanedioic acid (HDA) effectively stimulate respiration in state 4, and their action is, unlike the protonophore uncoupler DNP, is not caused by an increase in the proton conductivity of the inner membrane. Under these conditions, TMPD and HDA do not significantly affect the efficiency of oxidative ATP synthesis and energy transformation by complex IV (cytochrome c oxidase). The data obtained are considered as evidence that during the oxidation of succinate by liver mitochondria, TMPD and HDA selectively switch off ETC complex III from energy transformation. It is theoretically substantiated that, under these conditions, the H+/O ratio can be determined based on the ratio of respiratory rates in the absence and presence of TMPD and HDA. Based on this model, we considered the change in the H+/O ratio depending on the stimulation of mitochondrial respiration in state 4 by TMPD and HDA. It has been established that under the influence of TMPD or HDA, the value of the H+/O ratio decreases during the oxidation of succinate from 6 to the limiting values of 2. We conclude that in liver mitochondria during free respiration, in contrast to the oxidative ATP synthesis, the values of the H+/O ratio are 4 and 2 for complexes III and IV, respectively.

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Funding

The work was supported by grant from the Russian Foundation for Basic Research (20-015-00124).

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Authors and Affiliations

  1. Mari State University, 424001, Yoshkar-Ola, Russia

    V. N. Samartsev, A. A. Semenova, K. N. Belosludtsev & M. V. Dubinin

  2. Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, 142290, Pushchino, Russia

    K. N. Belosludtsev

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  1. V. N. Samartsev
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Correspondence to M. V. Dubinin.

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Statement on the welfare of animals. All procedures were performed in accordance with the European Communities Council Directive (November 24, 1986; 86/609/EEC) and the Declaration on humane treatment of animals. The Protocol of experiments was approved by the Commission on Bioethics of the Mari State University.

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Translated by A. Semenova

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Samartsev, V.N., Semenova, A.A., Belosludtsev, K.N. et al. Application of N,N,N',N'-Tetramethyl-p-Phenylenediamine and α,ω-Hexadecanedioic Acid for Determination of the H+/O Ratios of Complexes III and IV of the Liver Mitochondrial Respiratory Chain under Free Respiration Conditions. Biochem. Moscow Suppl. Ser. A 17, 117–126 (2023). https://doi.org/10.1134/S199074782302006X

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  • DOI: https://doi.org/10.1134/S199074782302006X

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