The Increase in the Peroxidase Activity of the Cytochrome with Substitutions in the Universal Binding Site Is Associated with Changes in the Ability to Interact with External Ligands.

Cytochrome (CytC), a one-electron carrier, transfers electrons from complex to cytochrome oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation.

Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties.

Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve this problem, a series of mutants were constructed with substitutions of Lys residues in the universal binding site (UBS) which interact electrostatically with negatively charged Asp and Glu residues at the binding sites of CytC partners, bc1 complex and CcO.

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.

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.

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

Bovine cytochrome c oxidase (CcO) contains two hemes, a and a, chemically identical but differing in coordination and spin state. The Soret absorption band of reduced aa-type cytochrome c oxidase consists of overlapping bands of the hemes a and a. It shows a peak at ∼444 nm and a distinct shoulder at ∼425 nm.

Direct Interaction of Mitochondrial Cytochrome 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 oxidase (CcO) mediated by direct contact with the enzyme.

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.

Interaction of Cytochrome C Oxidase with Steroid Hormones.

Estradiol, testosterone and other steroid hormones inhibit cytochrome oxidase (CcO) purified from bovine heart. The inhibition is strongly dependent on concentration of dodecyl-maltoside (DM) in the assay. The plots of K vs [DM] are linear for both estradiol and testosterone which may indicate an 1:1 stoichiometry competition between the hormones and the detergent.

Bicarbonate suppresses mitochondrial membrane depolarization induced by conventional uncouplers.

Bicarbonate has been known to modulate activities of various mitochondrial enzymes such as ATPase and soluble adenylyl cyclase. Here, we found that the ability of conventional protonophoric uncouplers, such as 2,4-dinitrophenol (DNP), carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), but not that of the new popular uncoupler BAM15, to decrease mitochondrial membrane potential was significantly diminished in the presence of millimolar concentrations of bicarbonate. Thus, the depolarizing activity of DNP and FCCP in mitochondria could be sensitive to the local concentration of bicarbonate in cells and tissues.

Effect of Ca on the redox potential of heme a in cytochrome c oxidase.

Subunit I of cytochrome c oxidase (CcO) from mitochondria and many bacteria contains a cation binding site (CBS) located at the outer positively charged aqueous phase not far from heme a. Binding of Ca with the CBS in bovine CcO inhibits activity of the enzyme 2-3 -fold [Vygodina, T., Kirichenko, A.

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 Ca and Na in a special cation binding site. Binding of Ca brings about partial inhibition of the enzyme while Na competes with Ca for the binding site and protects the enzyme from the inhibition [Vygodina, T., Kirichenko, A.

Calcium ions inhibit reduction of heme a in bovine cytochrome c oxidase.

The effect of Ca(2+) on the rate of heme a reduction by dithionite and hexaammineruthenium (RuAm) was studied in the cyanide-complexed bovine cytochrome oxidase (CcO). The rate of heme a reduction is proportional to RuAm concentration below 300 μM with kv of 0.53×10(6) M(-1) s(-1).

Cation binding site of cytochrome c oxidase: progress report.

Cytochrome c oxidase from bovine heart binds Ca(2+) reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+) shifts the absorption spectrum of heme a, which allowed earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A.

Reconstruction of absolute absorption spectrum of reduced heme a in cytochrome C oxidase from bovine heart.

This paper presents a new experimental approach for determining the individual optical characteristics of reduced heme a in bovine heart cytochrome c oxidase starting from a small selective shift of the heme a absorption spectrum induced by calcium ions. The difference spectrum induced by Ca2+ corresponds actually to a first derivative (differential) of the heme a(2+) absolute absorption spectrum. Such an absolute spectrum was obtained for the mixed-valence cyanide complex of cytochrome oxidase (a(2+)a3(3+)-CN) and was subsequently used as a basis spectrum for further procession and modeling.

Direct regulation of cytochrome c oxidase by calcium ions.

Cytochrome c oxidase from bovine heart binds Ca(2+) reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+) shifts the absorption spectrum of heme a, which allowed previously to determine the kinetics and equilibrium characteristics of the binding. However, no effect of Ca(2+) on the functional characteristics of cytochrome oxidase was revealed earlier.

Effect of calcium ions on electron transfer between hemes a and a(3) in cytochrome c oxidase.

Kinetics of the reduction of the hemes in cytochrome c oxidase in the presence of high concentration of ruthenium(III)hexaammine chloride was examined using a stopped-flow spectrophotometer. Upon mixing of the oxidized enzyme with dithionite and Ru(NH(3))(6)(3+), three well-resolved phases were observed: heme a reduction reaching completion within a few milliseconds is followed by two slow phases of heme a(3) reduction. The difference spectrum of heme a(3) reduction in the visible region is characterized by a maximum at ~612 nm, rather than at 603 nm as was believed earlier.

Catalase activity of cytochrome C oxidase assayed with hydrogen peroxide-sensitive electrode microsensor.

An iron-hexacyanide-covered microelectrode sensor has been used to continuously monitor the kinetics of hydrogen peroxide decomposition catalyzed by oxidized cytochrome oxidase. At cytochrome oxidase concentration ~1 µM, the catalase activity behaves as a first order process with respect to peroxide at concentrations up to ~300-400 µM and is fully blocked by heat inactivation of the enzyme. The catalase (or, rather, pseudocatalase) activity of bovine cytochrome oxidase is characterized by a second order rate constant of ~2·10(2) M(-1)·sec(-1) at pH 7.

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.

Peculiarities of cyanide binding to the ba3-type cytochrome oxidase from the thermophilic bacterium Thermus thermophilus.

Cytochrome c oxidase of the ba(3)-type from Thermus thermophilus does not interact with cyanide in the oxidized state and acquires the ability to bind heme iron ligands only upon reduction. Cyanide complexes of the reduced heme a(3) in cytochrome ba(3) and in mitochondrial aa(3)-type cytochrome oxidase are similar spectroscopically, but the a(3)(2+)-CN complex of cytochrome ba(3) is strikingly tight. Experiments have shown that the K(d) value of the cytochrome ba(3) complex with cyanide in the presence of reductants of the enzyme binuclear center does not exceed 10(-8) M, which is four to five orders of magnitude less than the K(d) of the cyanide complex of the reduced heme a(3) of mitochondrial cytochrome oxidase.

Inhibition of membrane-bound cytochrome c oxidase by zinc ions: high-affinity Zn2+-binding site at the P-side of the membrane.

In the presence of the uncoupler, external zinc ions inhibit rapidly turnover of cytochrome c oxidase reconstituted in phospholipid vesicles or bound to the membrane of intact mitochondria. The effect is promoted by electron leaks into the oxidase during preincubation with Zn(2+). Inhibition of liposome-bound bovine cytochrome oxidase by external Zn(2+) titrates with a K(i) of 1+/-0.

Peroxidase activity of mitochondrial cytochrome c oxidase.

Mitochondrial cytochrome c oxidase is able to oxidize various aromatic compounds like o-dianisidine, benzidine and its derivatives (diaminobenzidine, etc.), p-phenylenediamine, as well as amidopyrine, melatonin, and some other pharmacologically and physiologically active substances via the peroxidase, but not the oxidase mechanism. Although specific peroxidase activity of cytochrome c oxidase is low compared with classical peroxidases, its activity may be of physiological or pathophysiological significance due to the presence of rather high concentrations of this enzyme in all tissues, as well as specific localization of the enzyme in the mitochondrial membrane favoring accumulation of hydrophobic aromatic substances.

Cytochrome c oxidase as a calcium binding protein. Studies on the role of a conserved aspartate in helices XI-XII cytoplasmic loop in cation binding.

The aa(3)-type cytochrome c oxidases from mitochondria and bacteria contain a cation-binding site located in subunit I near heme a. In the oxidases from Paracoccus denitrificans or Rhodobacter sphaeroides, the site is occupied by tightly bound calcium, whereas the mitochondrial oxidase binds reversibly calcium or sodium that compete with each other. The functional role of the site has not yet been established.

Zinc ions as cytochrome C oxidase inhibitors: two sites of action.

Zinc ions are shown to be an efficient inhibitor of mitochondrial cytochrome c oxidase activity, both in the solubilized and the liposome-reconstituted enzyme. The effect of zinc is biphasic. First there occurs rapid interaction of zinc with the enzyme at a site exposed to the aqueous phase corresponding to the mitochondrial matrix.

Cytochrome c, an ideal antioxidant.

Generation of DeltaPsi (membrane potential) by cytochrome oxidase proteoliposomes oxidizing superoxide-reduced cytochrome c has been demonstrated. XO+HX (xanthine oxidase and hypoxanthine) were used to produce superoxide. It was found that the generation of DeltaPsi is completely abolished by cyanide (an uncoupler) or by superoxide dismutase, and is enhanced by nigericin.

Substitutions for glutamate 101 in subunit II of cytochrome c oxidase from Rhodobacter sphaeroides result in blocking the proton-conducting K-channel.

Two functional input pathways for protons have been characterized in the heme-copper oxidases: the D-channel and the K-channel. These two proton-conducting channels have different functional roles and have been defined both by X-ray crystallography and by the characterization of site-directed mutants. Whereas the entrance of the D-channel is well-defined as D132(I) (subunit I; Rhodobacter sphaeroides numbering), the entrance of the K-channel has not been clearly defined.