Requirement of Bacillus subtilis succinate:menaquinone oxidoreductase activity for membrane energization depends on the direction of catalysis.

Succinate:quinone oxidoreductases (SQR) from Bacilli catalyze reduction of menaquinone by succinate, as well as the reverse reaction. The direct activity is energetically unfavorable and lost upon ΔμН dissipation, thus suggesting ΔμН to be consumed during catalysis. Paradoxically, the generation of ΔμН upon fumarate reduction was never confirmed.

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.

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).

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.

Energization of Bacillus subtilis membrane vesicles increases catalytic activity of succinate:menaquinone oxidoreductase.

In this work, high DeltamicroH+-dependent succinate oxidase activity has been demonstrated for the first time with membrane vesicles isolated from Bacillus subtilis. The maximal specific rate of succinate oxidation by coupled inside-out membrane vesicles isolated from a B. subtilis strain overproducing succinate:menaquinone oxidoreductase approaches the specific rate observed with the intact cells.

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.

Stimulation of menaquinone-dependent electron transfer in the respiratory chain of Bacillus subtilis by membrane energization.

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A cytochrome bb'-type quinol oxidase in Bacillus subtilis strain 168.

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa(3) and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions.

Time-resolved generation of a membrane potential by ba3 cytochrome c oxidase from Thermus thermophilus. Evidence for reduction-induced opening of the binuclear center.

ba3-type cytochrome c oxidase purified from the thermophilic bacterium Thermus thermophilus has been reconstituted in phospholipid vesicles and laser flash-induced generation of a membrane potential by the enzyme has been studied in a microsecond/ms time scale with Ru(II)-tris-bipyridyl complex (RuBpy) as a photoreductant. Flash-induced single electron reduction of the aerobically oxidized ba3 by RuBpy results in two phases of membrane potential generation by the enzyme with tau values of about 20 and 300 microseconds at pH 8 and 23 degrees C. Spectrophotometric experiments show that oxidized ba3 reacts very poorly with hydrogen peroxide or any of the other exogenous heme iron ligands studied like cyanide, sulfide and azide.

Spontaneous spectral changes of the reduced cytochrome bd.

Reduction of the membrane-bound cytochrome bd from Bacillus subtilis, Escherichia coli and Azotobacter vinelandii as well as of the purified enzyme from E. coli was followed by secondary absorption changes on a time scale of tens of minutes. The difference absorption spectra of these changes resembled those induced by CO binding with heme d2+ indicating interaction of the heme with an endogenous pi-acceptor ligand.