Interaction of Terminal Oxidases with Amphipathic Molecules.

The review focuses on recent advances regarding the effects of natural and artificial amphipathic compounds on terminal oxidases. Terminal oxidases are fascinating biomolecular devices which couple the oxidation of respiratory substrates with generation of a proton motive force used by the cell for ATP production and other needs. The role of endogenous lipids in the enzyme structure and function is highlighted. The main regularities of the interaction between the most popular detergents and terminal oxidases of various types are described. A hypothesis about the physiological regulation of mitochondrial-type enzymes by lipid-soluble ligands is considered.

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

The Bile Acid Binding Site (BABS) of cytochrome oxidase (CcO) binds numerous amphipathic ligands. To determine which of the BABS-lining residues are critical for interaction, we used the peptide P4 and its derivatives A1-A4. P4 is composed of two flexibly bound modified α-helices from the M1 protein of the influenza virus, each containing a cholesterol-recognizing CRAC motif. The effect of the peptides on the activity of CcO was studied in solution and in membranes. The secondary structure of the peptides was examined by molecular dynamics, circular dichroism spectroscopy, and testing the ability to form membrane pores. P4 was found to suppress the oxidase but not the peroxidase activity of solubilized CcO. The Ki(app) is linearly dependent on the dodecyl-maltoside (DM) concentration, indicating that DM and P4 compete in a 1:1 ratio. The true Ki is 3 µM. The deoxycholate-induced increase in Ki(app) points to a competition between P4 and deoxycholate. A1 and A4 inhibit solubilized CcO with Ki(app)~20 µM at 1 mM DM. A2 and A3 hardly inhibit CcO either in solution or in membranes. The mitochondrial membrane-bound CcO retains sensitivity to P4 and A4 but acquires resistance to A1. We associate the inhibitory effect of P4 with its binding to BABS and dysfunction of the proton channel K. Trp residue is critical for inhibition. The resistance of the membrane-bound enzyme to inhibition may be due to the disordered secondary structure of the inhibitory peptide.

Individual heme a and heme a3 contributions to the Soret absorption spectrum of the reduced bovine cytochrome c oxidase.

Bovine cytochrome c oxidase (CcO) contains two hemes, a and a3, chemically identical but differing in coordination and spin state. The Soret absorption band of reduced aa3-type cytochrome c oxidase consists of overlapping bands of the hemes a2+ and a32+. It shows a peak at ∼444 nm and a distinct shoulder at ∼425 nm. However, attribution of individual spectral lineshapes to hemes a2+ and a32+ in the Soret is controversial. In the present work, we characterized spectral contributions of hemes a2+ and a32+ using two approaches. First, we reconstructed bovine CcO heme a2+ spectrum using a selective Ca2+-induced spectral shift of the heme a2+. Second, we investigated photobleaching of the reduced Thermus thermophilus ba3- and bovine aa3-oxidases in the Soret induced by femtosecond laser pulses in the Q-band. The resolved spectra show splitting of the electronic B0x-, B0y-transitions of both reduced hemes. The heme a2+ spectrum is shifted to the red relative to heme a32+ spectrum. The ∼425 nm shoulder is mostly attributed to heme a32+.

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

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.

Mechanism of Inhibition of Cytochrome c Oxidase by Triton X-100.

It is known that Triton X-100 (TX) reversibly inhibits activity of cytochrome c oxidase (CcO). The mechanism of inhibition is analyzed in this work. The action of TX is not directed to the reaction of CcO with cytochrome c, does not cause transition of the enzyme to the "slow" form, and is not associated with monomerization of the enzyme complex. TX completely suppresses oxygen reduction by CcO, but inhibition is prevented and partially reversed by dodecyl-ß-D-maltoside (DDM), a detergent used to maintain CcO in solution. A 1/1 stoichiometry competition is shown between DDM and TX for binding to CcO, with Ki = 0.3 mM and affinity of DDM for the enzyme of 1.2 mM. TX interaction with the oxidized enzyme induces spectral response with maximum at 421 nm and [TX]1/2 = 0.28 mM, presumably associated with heme a3. When CcO interacts with excess of H2O2 TX affects equilibrium of the oxygen intermediates of the catalytic center accelerating the FI-607 → FII-580 transition, inhibits generation of O2·- by the enzyme, and, to a lesser extent, suppresses the catalase partial activity. The observed effects can be explained by inhibition of the conversion of the intermediate FII-580 to the free oxidized state during the catalytic cycle. TX suppresses intraprotein electron transfer between hemes a and a3 during enzyme turnover. Partial peroxidase activity of CcO remains relatively resistant to TX under conditions that block oxidase reaction effectively. These features indicate an impairment of the K proton channel conductivity. We suggest that TX interacts with CcO at the Bile Acid Binding Site (BABS) that is located on the subunit I at the K-channel mouth and contacts with amphipathic regulators of CcO [Buhrow et al. (2013) Biochemistry, 52, 6995-7006]. Apparently, TX mimics the physiological ligand of BABS, whereas the DDM molecule mimics an endogenous phospholipid bound at the edge of BABS that controls effective affinity for the ligand.

Interaction of Cytochrome C Oxidase with Steroid Hormones.

Estradiol, testosterone and other steroid hormones inhibit cytochrome c oxidase (CcO) purified from bovine heart. The inhibition is strongly dependent on concentration of dodecyl-maltoside (DM) in the assay. The plots of Ki vs [DM] are linear for both estradiol and testosterone which may indicate an 1:1 stoichiometry competition between the hormones and the detergent. Binding of estradiol, but not of testosterone, brings about spectral shift of the oxidized CcO consistent with an effect on heme a33+. We presume that the hormones bind to CcO at the bile acid binding site described by Ferguson-Miller and collaborators. Estradiol is shown to inhibit intraprotein electron transfer between hemes a and a3. Notably, neither estradiol nor testosterone suppresses the peroxidase activity of CcO. Such a specific mode of action indicates that inhibition of CcO activity by the hormones is associated with impairing proton transfer via the K-proton channel.

Cytochrome c oxidase inhibition by calcium at physiological ionic composition of the medium: Implications for physiological significance of the effect.

Cytochrome c oxidase (CcO) from mammalian mitochondria binds Ca2+ and Na+ in a special cation binding site. Binding of Ca2+ brings about partial inhibition of the enzyme while Na+ competes with Ca2+ for the binding site and protects the enzyme from the inhibition [Vygodina, T., Kirichenko, A. and Konstantinov, A.A. (2013). Direct Regulation of Cytochrome c oxidase by Calcium Ions. PLoS One 8(9): e74436]. In the original studies, the inhibition was found to depend significantly on the ionic composition of the buffer. Here we describe inhibition of CcO by Ca2+ in media containing the main ionic components of cytoplasm (150mM KCl, 12mM NaCl and 1mM MgCl2). Under these conditions, Ca2+ inhibits CcO with effective Ki of 20-26µM, that is an order of magnitude higher than determined earlier in the absence of Na+. At physiological value of ionic strength, the inhibition can be observed at any turnover number of CcO, rather than only at low TN (<10s-1) as found previously. The inhibition requires partially oxidized state of cytochrome c and is favored by high ionic strength with a sharp transition at 0.1-0.2M. The high Ki=20-26µM found for CcO inhibition by calcium matches closely the known value of "Km" for Ca2+-induced activation of the mitochondrial calcium uniporter. The inhibition of CcO by Ca2+ is proposed to modulate mitochondrial Ca2+-uptake via the mitochondrial calcium uniporter, promote permeability transition pore opening and induce reduction of Mia40 in the mitochondrial intermembrane space.

Circular dichroism spectra of cytochrome c oxidase.

Circular dichroism spectra of bovine heart aa(3)-type cytochrome c oxidase have been studied with a major focus on the Soret band π → π* transitions, B(0(x,y)), in the two iron porphyrin groups of the enzyme. The spectra of the fully reduced and fully oxidized enzyme as well as of its carbon monoxide and cyanide complexes have been explored. In addition, CD spectra of the reduced and oxidized ba(3)-type cytochrome c oxidase from Thermus thermophilus were recorded for comparison. An attempt is made to interpret the CD spectra of cytochrome c oxidase with the aid of a classical model of dipole-dipole coupled oscillators taking advantage of the known 3D crystal structure of the enzyme. Simultaneous modeling of the CD and absorption spectra shows that in the bovine oxidase, the dipole-dipole interactions between the hemes a and a(3), although contributing significantly, cannot account either for the lineshape or the magnitude of the experimental spectra. However, adding the interactions of the hemes with 22 aromatic amino acid residues located within 12 Å from either of the two heme groups can be used to model the CD curves for the fully reduced and fully oxidized oxidase with reasonable accuracy. Interaction of the hemes with the peptide bond transition dipoles is found to be insignificant. The modeling indicates that the CD spectra of cytochrome oxidase in both the reduced and oxidized states are influenced significantly by interaction with Tyr244 in the oxygen-reducing center of the enzyme. Hence, CD spectroscopy may provide a useful tool for monitoring the redox/ionization state of this residue. The modeling confirms wide energy splitting of the orthogonal B(x) and B(y) transitions in the porphyrin ring of heme a.