Alexander V. Bogachev

Aeration-dependent changes in composition of the quinone pool in Escherichia coli: Evidence of post-transcriptional regulation of the quinone biosynthesis

© 1997 Federation of European Biochemical Societies.

The origin of the sodium-dependent NADH oxidation by the respiratory chain of Klebsiella pneumoniae

Properties of Klebsiella pneumoniae respiratory chain enzymes catalyzing NADH oxidation have been studied. Using constructed K. pneumoniae mutant strains, it was shown that three enzymes belonging to different families of NADH:quinone oxidoreductases operate in this bacterium. The NDH‐2‐type enzyme is not coupled with energy conservation, the NDH‐1‐type enzyme is a primary proton pump, and the NQR‐type enzyme is homologous to the sodium‐motive NADH dehydrogenase of Vibrio and is shown to be a primary Na+ pump. It is concluded that the NQR‐type enzyme, not the NDH‐1‐type enzyme, catalyzes sodium‐dependent NADH oxidation in K. pneumoniae.

Sodium-translocating NADH:quinone oxidoreductase as a redox-driven ion pump.

The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) is a component of the respiratory chain of various bacteria. This enzyme is an analogous but not homologous counterpart of mitochondrial Complex I. Na+-NQR drives the same chemistry and also uses released energy to translocate ions across the membrane, but it pumps Na+ instead of H+. Most likely the mechanism of sodium pumping is quite different from that of proton pumping (for example, it could not accommodate the Grotthuss mechanism of ion movement); this is why the enzyme structure, subunits and prosthetic groups are completely special. This review summarizes modern knowledge on the structural and catalytic properties of bacterial Na+-translocating NADH:quinone oxidoreductases. The sequence of electron transfer through the enzyme cofactors and thermodynamic properties of those cofactors is discussed. The resolution of the intermediates of the catalytic cycle and localization of sodium-dependent steps are combined in a possible molecular mechanism of sodium transfer by the enzyme.

Na(+)-Translocating NADH:quinone oxidoreductase: progress achieved and prospects of investigations.

Structural and catalytic properties of bacterial Na+-translocating NADH: quinone oxidoreductases are briefly described. Special attention is given to studies on kinetics of the enzyme interaction with NADH and the role of sodium ions in this process. Based on the existing data, possible model mechanisms of sodium transfer by Na+-translocating NADH:quinone oxidoreductase are proposed.

Functional analysis of the Na+/H+ antiporter encoding genes of the cyanobacterium Synechocystis PCC 6803

The role of putative Na+/H+ antiporters encoded by nhaS1 (slr1727), nhaS3 (sll0689), nhaS4 (slr1595), and nhaS5 (slr0415) in salt stress response and internal pH regulation of the cyanobacterium Synechocystis PCC 6803 was investigated. For this purpose the mutants (single, double, and triple) impaired in genes coding for Na+/H+ antiporters were constructed using the method of interposon mutagenesis. PCR analyses of DNA demonstrated that mutations in nhaS1, nhaS4, and nhaS5 genes were segregated completely and the mutants contained only inactivated copies of the corresponding genes. Na+/H+ antiporter encoded by nhaS3 was essential for viability of Synechocystis since no completely segregated mutants were obtained. The steady-state intracellular sodium concentration and Na+/H+ antiporter activities were found to be the same in the wild type and all mutants. No differences were found in the growth rates of wild type and mutants during their cultivation in liquid media supplemented with 0.68 M or 0.85 M NaCl as well as in media buffered at pH 7.0, 8.0, or 9.0. The expression of genes coding for Na+/H+ antiporters was studied. No induction of any Na+/H+ antiporter encoding gene expression was found in wild type or single mutant cells grown under high salt or at different pH values. Nevertheless, in cells of double and triple mutants adapted to high salt or alkaline pH some of the remaining Na+/H+ antiporter encoding genes showed induction. These results might indicate that some of Na+/H+ antiporters can functionally replace each other under stress conditions in Synechocystis cells lacking the activity of more than one antiporter.

Noncoupled NADH:Ubiquinone Oxidoreductase of Azotobacter vinelandii Is Required for Diazotrophic Growth at High Oxygen Concentrations

The gene encoding the noncoupled NADH:ubiquinone oxidoreductase (NDH II) from Azotobacter vinelandii was cloned, sequenced, and used to construct an NDH II-deficient mutant strain. Compared to the wild type, this strain showed a marked decrease in respiratory activity. It was unable to grow diazotrophically at high aeration, while it was fully capable of growth at low aeration or in the presence of NH(4)(+). This result suggests that the role of NDH II is as a vital component of the respiratory protection mechanism of the nitrogenase complex in A. vinelandii. It was also found that the oxidation of NADPH in the A. vinelandii respiratory chain is catalyzed solely by NDH II.

Involvement of a d-type oxidase in the Na+-motive respiratory chain of Escherichia coli growing under low Δ\̄gmH+ conditions

An attempt has been made to find out which of the two terminal oxidases, the d‐type or the o‐type, operates as a Na+ pump in Escherichia coli grown at low Δ\̄gmH+ conditions. For this purpose, mutants lacking either d or o oxidase have been studied. It is shown that a d −,o + mutant grows slowly or does not grow at all under low Δ\̄gmH+ conditions (alkaline or protonophore‐containing growth media were used). Inside‐out subcellular vesicles from the d −,o + mutant cannot oxidize ascorbate and TMPD, and cannot transport Na+ when succinate is oxidize in the presence of a protonophore. The same vesicles are found to transport Na+ when NADH is oxidized as if the Na+‐motive NADH‐quinone oxidase were operative. On the other hand, a mutant lacking o oxidase (d +,o −) grows at low Δ\̄gmH+ conditions as fast as the maternal E. coli strain containing both d and o oxidases. Corresponding vesicles oxidize ascorbate and TMPD as well as succinate, the oxidations being coupled to the protonophore‐stimulated Na+ transport. Growth in the presence of a protonophore is found to induce a strong increase in the d oxidase level in the maternal d +,o + E. coli strain. It is concluded that oxidase of the d‐type, rather than of the o‐type, operates as a Na+ pump in E. coli grown under conditions unfavorable for the H+ cycle.

Cytochrome d induction in Escherichia coli growing under unfavorable conditions

Growth of E. Coli in the presence of the protonophorous uncoupler pentachlorophenol is shown to strongly enhance levels of cytochrome d, a putative Na+‐motive oxidase. This effect was found to be arrested by chloramphenicol and stimulated by high Na+ concentration in the growth medium. The induction of cytochrome d takes place in a mutant deficient in the F0F1 ATP‐synthase but does not occur in mutants deficient in either of two different components of the Arc system. Similar relationships were revealed when pentachlorophenol was replaced by ferricyanide and phenazine methosulfate, agents oxidizing the respiratory chain. Induction of cytochrome d is also shown to occur in riboflavin‐deficient mutants growing in the presence of such low riboflavin concentrations as to be insufficient to maintain a high respiration rate. It is suggested (i) that it is Δ−μH+ decrease rather than reduction of the respiratory chain that is the signal for the induction of cytochrome d, and (ii) the Arc system is involved in this type of metabolic regulation.

The Na+/e stoichiometry of the Na+-motive NADH : quinone oxidoreductase in Vibrio alginolyticus

A method is proposed to estimate the stoichiometries of primary Na+‐pumps in intact bacterial cells. It is based on technique when the H+/e− stoichiometry is measured in the presence of protophorous uncoupler and in the absence of penetrating ions other than H+. Under these conditions, the H+ influx discharges membrane potential generated by the Na+ pump so the Na+/e− and H+/e− ratios become equal. Using this approach it is shown that the Na+/e− ratio for the Na+‐motive NADH : quinone oxidoreductase of Vibrio alginolyticus is equal to 0.71±0.06. The Na+/e− stoichiometry appears to be ≈1, provided that the contribution of the non‐coupled NADH : quinone oxidoreductase, which is resistant to low HQNO concentrations, is taken into account.

Kinetics of the spectral changes during reduction of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi

Two radical signals with different line widths are seen in the Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi by EPR spectroscopy. The first radical is observed in the oxidized enzyme, and is assigned as a neutral flavosemiquinone. The second radical is observed in the reduced enzyme and is assigned to be the anionic form of flavosemiquinone. The time course of Na+-NQR reduction by NADH, as monitored by stopped-flow optical spectroscopy, shows three distinct phases, the spectra of which suggest that they correspond to the reduction of three different flavin species. The first phase is fast both in the presence and absence of sodium, and is assigned to reduction of FAD to FADH2 at the NADH dehydrogenating site. The rates of the other two phases are strongly dependent on sodium concentration, and these phases are attributed to reduction of two covalently bound FMNs. Combination of the optical and EPR data suggests that a neutral FMN flavosemiquinone preexists in the oxidized enzyme, and that it is reduced to the fully reduced flavin by NADH. The other FMN moiety is initially oxidized, and is reduced to the anionic flavosemiquinone. One-electron transitions of two discrete flavin species are thus assigned as sodium-dependent steps in the catalytic cycle of Na+-NQR.