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Direct Interaction of Mitochondrial Cytochrome c Oxidase with Thyroid Hormones: Evidence for Two Binding Sites
Ilya P Oleynikov1, Roman V Sudakov2, Natalia V Azarkina1
1A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, Bld. 40, 119992 Moscow, Russia.
Abstract:
Thyroid hormones regulate tissue metabolism to establish an energy balance in the cell, in particular, by affecting oxidative phosphorylation. Their long-term impact is mainly associated with changes in gene expression, while the short-term effects may differ in their mechanisms. Our work was devoted to studying the short-term effects of hormones T2, T3 and T4 on mitochondrial cytochrome c oxidase (CcO) mediated by direct contact with the enzyme. The data obtained indicate the existence of two separate sites of CcO interaction with thyroid hormones, differing in their location, affinity and specificity to hormone binding. First, we show that T3 and T4 but not T2 inhibit the oxidase activity of CcO in solution and on membrane preparations with Ki ≈ 100-200 μM. In solution, T3 and T4 compete in a 1:1 ratio with the detergent dodecyl-maltoside to bind to the enzyme. The peroxidase and catalase partial activities of CcO are not sensitive to hormones, but electron transfer from heme a to the oxidized binuclear center is affected. We believe that T3 and T4 could be ligands of the bile acid-binding site found in the 3D structure of CcO by Ferguson-Miller's group, and hormone-induced inhibition is associated with dysfunction of the K-proton channel. A possible role of this interaction in the physiological regulation of the enzyme is discussed. Second, we find that T2, T3, and T4 inhibit superoxide generation by oxidized CcO in the presence of excess H2O2. Inhibition is characterized by Ki values of 0.3-5 μM and apparently affects the formation of O2●- at the protein surface. The second binding site for thyroid hormones presumably coincides with the point of tight T2 binding on the Va subunit described in the literature.
Insights
Thyroid hormones T3 and T4 directly interact with cytochrome c oxidase (CcO), inhibiting its activity and superoxide generation through two distinct binding sites. This study reveals short-term regulatory mechanisms of thyroid hormones on cellular energy balance.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Metabolism
Background:
- Thyroid hormones (T2, T3, T4) regulate cellular metabolism and energy balance, primarily through effects on oxidative phosphorylation.
- While long-term effects involve gene expression, short-term mechanisms of thyroid hormone action on mitochondrial enzymes remain less understood.
Purpose of the Study:
- To investigate the short-term effects of thyroid hormones (T2, T3, T4) on mitochondrial cytochrome c oxidase (CcO) activity.
- To identify potential direct interaction sites and mechanisms of thyroid hormone binding to CcO.
Main Methods:
- Enzyme kinetics assays were performed to measure CcO activity in solution and membrane preparations.
- Competitive binding studies using dodecyl-maltoside were conducted to characterize hormone interaction.
- Superoxide generation assays were used to assess the effect of hormones on CcO's reactive oxygen species production.
Main Results:
- Thyroid hormones T3 and T4 inhibit CcO oxidase activity (Ki ≈ 100-200 μM) by binding to a site overlapping with the bile acid-binding site, affecting electron transfer.
- T3 and T4 compete with dodecyl-maltoside for binding, suggesting interaction at the detergent-binding site.
- T2, T3, and T4 inhibit CcO-mediated superoxide generation (Ki ≈ 0.3-5 μM) at a distinct site on the protein surface, potentially involving the Va subunit.
Conclusions:
- Cytochrome c oxidase (CcO) possesses at least two distinct thyroid hormone binding sites, mediating different inhibitory effects on enzyme activity and function.
- These findings suggest a direct, short-term regulatory role for thyroid hormones in modulating mitochondrial oxidative phosphorylation and reactive oxygen species production.
- The identified binding sites and mechanisms provide new insights into the physiological regulation of CcO by thyroid hormones.
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