Direct Interaction of Mitochondrial Cytochrome c Oxidase with Thyroid Hormones: Evidence for Two Binding Sites

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.

Structural basis of mammalian complex IV inhibition by steroids

“…The absorbance spectra of the redox cofactors in CIV change with redox state, making it possible to determine how electrons are distributed among the internal electron carriers. Results from earlier stopped-flow measurements with R. sphaeroides CIV revealed that bile salts caused accumulation of electrons at heme a , suggesting that the electron transfer from heme a to the catalytic site is rate limiting ( 21 , 23 , 36 ).…”

“…Lipophilic small molecules, including detergents, steroids, and bile salts, have a complex effect on the activity of CIV from Rhodobacter sphaeroides ( 18 , 21 ). Steroids as well as thyroid hormones have been shown to inhibit mammalian CIV ( 22 , 23 ), possibly by interfering with K pathway proton translocation ( 24 ), and thyroid hormones also appear to suppress superoxide formation by CIV ( 23 ). This effect has also been seen when mitochondria are exposed to steroids and glucocorticoids, which causes decreased oxygen consumption, an effect that is being explored in relation to cancer therapies ( 25 , 26 ).…”

Bioenergetic Aspects of Mitochondrial Actions of Thyroid Hormones

“…Of note, these last-mentioned studies preceded subsequent ones that revealed the existence, in COX, of a steroid binding site conserved from Rhodobacter sphaeroides to mammals with high affinity for the bile salt deoxycholate [121], paving the way for the conceptualization of a COX regulatory site for steroids. Indeed, more recently, estradiol, testosterone, other steroids and THs have been shown to bind to and inhibit bovine heart purified COX [122,123]. Specifically, as far as it concerns THs, the existence of two separate sites of COX interaction, differing in location, affinity and specificity to hormone binding, has been reported [123], one of the two presumably being the point of T2 binding on the Va subunit described in [119].…”

“…Indeed, more recently, estradiol, testosterone, other steroids and THs have been shown to bind to and inhibit bovine heart purified COX [122,123]. Specifically, as far as it concerns THs, the existence of two separate sites of COX interaction, differing in location, affinity and specificity to hormone binding, has been reported [123], one of the two presumably being the point of T2 binding on the Va subunit described in [119].…”

Interaction of Amphipathic Peptide from Influenza Virus M1 Protein with Mitochondrial Cytochrome Oxidase

“…Recent studies of our laboratory on mitochondrial CcO revealed that some steroids and secosteroids [ 47 ], the steroid-mimicking detergent Triton X-100 [ 48 ], and thyroid hormones [ 49 ] bind to BABS inducing effective enzyme inhibition. Such a wide specificity of the site brings us to the assumption that its true physiological ligand is still unknown.…”