Identification of Na+-Pumping Cytochrome Oxidase in the Membranes of Extremely Alkaliphilic Thioalkalivibrio Bacteria.

For the first time, the functioning of the oxygen reductase Na+-pump (Na+-pumping cytochrome c oxidase of the cbb3-type) was demonstrated by examining the respiratory chain of the extremely alkaliphilic bacterium Thioalkalivibrio versutus [Muntyan, M. S., et al. (2015) Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase, Proc. Natl. Acad. Sci. USA, 112, 7695-7700], a product of the ccoNOQP operon. In this study, we detected and identified this enzyme using rabbit polyclonal antibody against the predicted C-terminal amino acid sequence of its catalytic subunit. We found that this cbb3-type oxidase is synthesized in bacterial cells, where it is located in the membranes. The 48-kDa oxidase subunit (CcoN) is catalytic, while subunits CcoO and CcoP with molecular masses of 29 and 34 kDa, respectively, are cytochromes c. The theoretical pI values of the CcoN, CcoO, and CcoP subunits were determined. It was shown that parts of the CcoO and CcoP subunits exposed to the aqueous phase on the cytoplasmic membrane P-side are enriched with negatively charged amino acid residues, in contrast to the parts of the integral subunit CcoN adjacent to the aqueous phase. Thus, the Na+-pumping cytochrome c oxidase of T. versutus, both in function and in structure, demonstrates adaptation to extremely alkaline conditions.

Evaluation of the electrical potential on the membrane of the extremely alkaliphilic bacterium Thioalkalivibrio.

The electrical potential on the membrane was measured in cells of strains AL2 and ALJ15 of the extremely alkaliphilic bacterium Thioalkalivibrio versutus using the penetrating cation tetraphenylphosphonium (TPP(+)) and a TPP(+)-selective electrode. The potentials were -228 ± 5 and -224 ± 5 mV, respectively, i.e. higher than in most alkaliphilic bacteria. Membrane potential in the cells was estimated by measuring the inner cell volume by two independent methods: (1) estimation of total cell volume by light microscopy and (2) estimation of the inner aqueous volume of the cells with allowance for the distribution difference of tritium labeled water penetrating through the membranes and a nonpenetrating colored protein. The inner cell volume was 2.4 ± 0.2 and 2.2 ± 0.1 µl/mg of cell protein by the two methods, respectively. Computer computation was used as an alternative to manual calculation to count the number of cells for estimation of total cell volume.

Study of redox potential in cytochrome c covalently bound to terminal oxidase of alkaliphilic Bacillus pseudofirmus FTU.

Spectroelectrochemistry was used to determine the midpoint redox potentials of heme cofactors of the caa3-type cytochrome oxidase from the alkaliphilic bacterium Bacillus pseudofirmus FTU. The apparent midpoint potentials (E(m)(app)) for the most prominent transitions of hemes a and a3 (+193 and +334 mV, respectively) were found to be similar to the values reported for other enzymes with high homology to the caa3-type oxidase. In contrast, the midpoint potential of the covalently bound cytochrome c (+89 mV) was 150-170 mV lower than in cytochromes c, either low molecular weight or covalently bound to the caa3 complex in all known aerobic neutralophilic and thermo-neutralophilic bacteria. Such an unusually low redox potential of the covalently bound cytochrome c of the caa3-type oxidase of alkaliphilic bacteria, together with high redox potentials of hemes a and a3, ensures more than twice higher difference in redox potentials inside the respiratory complex compared to the homologous mitochondrial enzyme. The energy released during this redox transition might be stored in the transmembrane H+ gradient even under low Deltap in the alkaline environment of the bacteria at the expense of a significant increase in DeltaG of the coupled redox reaction.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 1. Cationic plastoquinone derivatives: synthesis and in vitro studies.

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the "window" between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH2, the rate of oxidation of SkQH2 being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH*. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C(1/2) values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1x10(-11) and 8x10(-13) M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Deltapsi values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000 : 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3x10(8) times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo.

Energetics of alkalophilic representatives of the genus Bacillus.

Cytochrome and lipid composition of membranes is considered as the attributes required for adaptation of the alkalophiles to alkaline conditions. Respiratory chains of alkalophilic representatives of the genus Bacillus are discussed. Special attention is paid to the features of the Na(+)-cycle of these bacteria and to the features determining halo- and alkalotolerant phenotype, which have been reported due to recent achievements in genomics.

Uncoupling effect of fatty acids in halo- and alkalotolerant bacterium Bacillus pseudofirmus FTU.

Natural uncouplers of oxidative phosphorylation, long-chain non-esterified fatty acids, cause uncoupling in the alkalo- and halotolerant bacterium Bacillus pseudofirmus FTU. The uncoupling effect in the bacterial cells was manifested as decrease of membrane potential and increase of respiratory activity. The membrane potential decrease was detected only in bacterial cells exhausted by their endogenous substrates. In proteoliposomes containing reconstituted bacterial cytochrome c oxidase, fatty acids caused a "mild" uncoupling effect by reducing membrane potential only at low rate of membrane potential generation. "Free respiration" induced by the "mild" uncouplers, the fatty acids, can be considered as possible mechanism responsible for adaptation of the bacteria to a constantly changed environment.

Ion transport coupled to terminal oxidase functioning in the extremely alkaliphilic halotolerant bacterium Thioalkalivibrio.

Proton transport in the terminal part of the respiratory chain in the extremely alkaliphilic halotolerant bacterial strain Thioalkalivibrio versutus was studied under near-optimum growth conditions (pH 9.0-9.5). Under these conditions, bacterial cells generated electric potential with the negative charge being inside the cells. When only the terminal part of the respiratory chain functioned, it was found that: 1) unlike other bacteria known, this bacterium did not acidify the medium in the presence of K(+) and valinomycin; 2) in the presence of an uncoupler, CCCP, but in the absence of valinomycin, reversible alkalinization of the medium occurred as a result of proton influx into the cells. Cyanide prevented this alkalinization. The difference spectra indicate that cell membranes contained cytochromes c and (b+o), some of which reacted with CO. The respiratory activity of membranes in the terminal part of the respiratory chain was optimal at pH 9.5 and specifically depended on sodium ions (C(1/2) = 10 mM). The data suggest the presence of a Na(+)-pump in the terminal part of the respiratory chain of the studied strain which can pump Na(+) out of the cells.

Lithoautotrophic growth of the freshwater strain Beggiatoa D-402 and energy conservation in a homogeneous culture under microoxic conditions.

The freshwater filamentous bacterium Beggiatoa D-402 was shown to grow lithoautotrophically in a homogeneous culture under microoxic conditions only, the growth yield being the highest at 0.1 mg O(2) l(-1). High activities of the Calvin cycle key enzymes and of the dissimilatory path thiosulfate oxidation enzymes were found in the bacterial cells. The rate of CO(2) fixation above 112 nmol min(-1) (mg protein)(-1), an about 90% increase in the protein carbon at the expense of CO(2) carbon and an increase in the molar yield up to 12 mg dry weight (mmol oxidized thiosulfate)(-1) indicate the bacterial growth was autotrophic. Thiosulfate was oxidized by the strain almost completely into sulfate. The metabolically useful energy was conserved by oxidative phosphorylation that was coupled to oxidation of sulfur compounds. The bacterial membranes were found to contain CO-binding cytochromes b and two cytochromes c with M(r) 23 and 26 kDa, the terminal part of the respiratory chain containing presumably a cbb(3)-type oxidase. A cytochrome c with M(r) 12 kDa was detected in the soluble fraction.

Transfer of cationic antibacterial agents berberine, palmatine, and benzalkonium through bimolecular planar phospholipid film and Staphylococcus aureus membrane.

Some organic cations are known to be electrophoretically imported into bacterial cells and actively extruded from these cells by multidrug resistance (MDR) pumps. We have studied penetration of plant antimicrobial agents berberine and palmatine and synthetic antiseptic benzalkonium chloride through black planar phospholipid membrane (BLM) and membrane of Staphylococcus aureus cells. Gradients of these cations across BLM generated an electric potential difference. Penetrating anion tetraphenyl borate and phloretin (a plant substance decreasing membrane dipole potential) stimulated this effect. Under optimal conditions, the magnitude of the electric potential was close to theoretical, that is, 60 mV/10-fold cation gradient. Berberine accumulated in S. aureus cells as shown by direct measurement of berberine with a berberine-sensitive electrode. The berberine accumulation was prevented by protonophore CCCP and was stimulated by mutation in the MDR pump NorA. It is concluded that the plant alkaloids and benzalkonium are penetrating cations and substrates of an MDR pump.

Role of copper during carbon monoxide binding to terminal oxidases.

Under fully reduced conditions, reassociation kinetics of CO were studied in several terminal oxidases containing copper in their binuclear center. The purified Paracoccus denitrificans ba3-type quinol oxidase was found to recombine with CO monophasically (tau 25-30 ms) like oxidases of the bo type from Escherichia coli, the caa3 type from Bacillus halodurans FTU, and the bo type from Methylobacillus flagellatum KT. Oxidase of the aa3 type from bovine heart recombined with CO monophasically at a higher rate (tau 16-19 ms) than the studied copper-containing bacterial oxidases. After prolonged incubation in the presence of CO, oxidases of the ba3 and aa3 types changed their CO-binding properties. The contribution of the slow component was diminished while new fast components arose. Measurement of the metal content in the oxidases indicated that during the incubation, the enzymes lost their copper, the process being accompanied by the appearance of a fast CO recombination rate resembling that of the non-copper oxidases of the bd type from E. coli and the bb type from Bacillus halodurans FTU. This points to a role of copper in CO binding by terminal oxidases.

Identification of Bacillus sp. FTU strain and the study of the caa3-type oxidase homology.

The culture, morphology, and genome of alkalo- and halotolerant bacterial strain Bacillus sp. FTU were characterized; the strain is compared to other representatives of genus Bacillus. The DNA-DNA hybridization data indicate that the strains of Bacillus halodurans DSM 497 and DSM 2513 and Bacillus sp. FTU belong to the same species. Bacillus sp. FTU can be renamed to Bacillus halodurans FTU. The N-terminal amino acid fragments of the subunits I and II of the terminal caa3-type cytochrome c oxidase of B. halodurans FTU were sequenced. The N-terminal fragments of this enzyme and of the caa3-type oxidase of alkalophilic Bacillus firmus OF4 are highly homologous (homology of subunits I and II is over 90 and over 96%, respectively). Such high homology of the terminal oxidases of these bacteria might be due to their alkaline medium.

Terminal oxidases of the bb- and caa3-types in Bacillus sp. FTU.

We previously identified two oxidases in the membranes of bacterium Bacillus sp. FTU. One of them slowly (caa3) and the other rapidly (bo) recombines with carbon monoxide (CO) after laser flash photolysis, in this respect resembling the Escherichia coli bo- and bd-type oxidases, respectively. In the present study we found three copper atoms in the slowly CO-recombining oxidase from Bacillus sp. FTU. In the other oxidase, the copper content is very low and clearly substoichiometric. Reversed-phase chromatography revealed the presence of haems A and C in the Bacillus sp. FTU copper-containing oxidase and haems B and C in the non-copper-containing one. We thus suggest that the Bacillus sp. FTU oxidase rapidly reacting with CO previously attributed to bo-type by analogy in redox spectrum with the E. coli enzyme be redefined as bb-type oxidase.

Two o-type oxidases in Methylobacillus flagellatum KT.

Two oxidases of the o-type in membranes of the methanol-grown obligate methylotroph Methylobacillus flagellatum KT were distinguished. For this purpose the kinetic analysis of the laser flash-induced optical absorbance changes of CO-oxidase complexes under reducing conditions was used. The ratio of these oxidases in membranes greatly depended on the phases of bacterial growth. One of the oxidases appeared to belong to the Escherichia coli o-type oxidase family being more sensitive to KCN (Ki = 1 microM). It showed monophasic CO recombination kinetics with tau 25-30 ms and was expressed in the early exponential phase of growth. The other oxidase seemed to be similar to the Bacillus sp. FTU o-type oxidase being less sensitive to KCN (Ki = 6 microM), having three-phasic CO reassociation kinetics with tau 35-70 microseconds, 0.25-0.5 ms and 2-4 ms and dominating in the stationary growth phase. Pyridine haemochrome spectra showed haems A and D to be absent from the bacterial membranes.

Kinetics of CO binding to putative Na(+)-motive oxidases of the o-type from Bacillus FTU and of the d-type from Escherichia coli.

The kinetics of CO reassociation with isolated Bacillus FTU o-type oxidase and with solubilized membranes of Escherichia coli (GO102 strain) containing the d-type oxidase only, upon laser flash photolysis under reducing conditions, were studied. In both cases, kinetics are shown to be composed of three phases (tau 35-70 microseconds, 0.25-0.5 ms and 2-5 ms). The spectra of the flash-induced absorbance changes of the first kinetic components proved to be characteristic of CO-o- and CO-b595 d-cytochrome complexes in Bac. FTU and E. coli, respectively. The spectra of the second and the third components appeared to be nearly the same in Bac. FTU and E. coli with peaks for the former at 436-437 and 590 nm and troughs at 419-420 and 569 nm; and for the latter with peaks at 436-437 and 558-560 nm and troughs at 419-420 and 575-578 nm. The similarity between the putative Na(+)-pumping Bac. FTU o- and E. coli d-type oxidases and their difference from the H(+)-motive Bac. FTU caa3- and E. coli o-type oxidases are discussed.

Kinetics of CO binding to H(+)-motive oxidases of the caa3-type from Bacillus FTU and of the o-type from Escherichia coli.

The kinetics of CO rebinding with isolated Bacillus FTU caa3-type oxidase and with solubilized Escherichia coli membranes (GO103 strain) containing the o-type oxidase as the main O2-reducing enzyme were studied under reducing conditions by laser flash photolysis of the CO-oxidase complexes. The spectra of the optical absorbance changes upon photolysis were characteristic of CO-caa3- and CO-o-oxidase complexes in Bac. FTU and E. coli, respectively. Small quantities of d-type oxidase in E. coli GO103 membranes were detected. The kinetics of CO reassociation with reduced caa3- and o-type oxidases were monophasic with tau 25-30 ms in both cases.

The F1-type ATPase in anaerobic Lactobacillus casei.

An ATPase from anaerobic Lactobacillus casei has been isolated and 100-times purified. The 400 kDa enzyme molecule was found to have a hexagonal structure 10 nm in diameter composed of at least six protein masses. SDS-electrophoresis reveals four or, under certain conditions, five types of subunit, of apparent molecular masses 57 (alpha), 55 (beta), 40 (gamma), 22 (delta) and 14 (epsilon) kDa with stoichiometry of 3 alpha, 3 beta, gamma, delta, epsilon. The following features resembling F1-ATPases from other sources were found to be inherent in the solubilized L. casei ATPase. (i) Detachment from the membrane desensitizes ATPase to low DCCD concentrations and sensitizes it to water-soluble carbodiimide. (ii) Soluble ATPase is inhibited by Nbf chloride and azide, is resistant to SH-modifiers and is activated by sulfite and octyl glucoside, the activating effect being much stronger than in the case of the membrane-bound ATPase. Substrate specificity of the enzyme is also similar to that of other factors F1. Divalent cations strongly activate the soluble enzyme when added at a concentration equal to that of ATP. An excess of Mn2+, Mg2+ or Co2+ inhibits ATPase activity of F1, whereas that of Ca2+ induces its further activation. No other F1-like ATPases are found in L. casei. It is concluded that this anaerobic bacterium possesses a typical F1-ATPase similar to those in mitochondria, chloroplasts, aerobic and photosynthetic eubacteria.