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.

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.

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.

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.

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.