Yulia V. Bertsova

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.

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.

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.

Generation of protonic potential by the bd-type quinol oxidase of Azotobacter vinelandii.

Inside‐out subcellular vesicles of Azotobacter vinelandii are found to produce ΔpH and ΔΨ (interior acidic and positive) when oxidising malate or menadiol. These effects are inherent in both Cyd+Cyo− (lacking the o‐type oxidase) and Cyd+Cyo− (lacking the bd‐type oxidase) strains. They appear to be myxothiazol‐sensitive in the Cyd−Cyo+ strain but not in the Cyd+Cyo− strain. The H+/e− ratio for the terminal part of respiratory chain of a bd‐type oxidase overproducing strain is established as being close to 1. It is also shown that NADH oxidation by the vesicles from the Cyd−Cyo+ strain is sensitive to low concentrations of myxothiazol and antimycin A whereas that of the Cyd+Cyo− strain is resistant to these Q‐cycle inhibitors. It is concluded that (i) the bd‐type oxidase of A. vinelandii is competent in generating a protonic potential but its efficiency is lower than that of the o‐type oxidase and (iii) Q‐cycle does operate in the o‐type cytochrome oxidase terminated branch of the A. vinelandii respiratory chain and does not in the bd‐type quinol oxidase terminated branch. These relationships are discussed in the context of the respiratory protection function of the bd‐type oxidase in A. vinelandii.

Alternative pyrimidine biosynthesis protein ApbE is a flavin transferase catalyzing covalent attachment of FMN to a threonine residue in bacterial flavoproteins

Background: The ApbE protein with unknown function is widespread in bacteria. Results: ApbE catalyzes Mg2+-dependent FMN transfer from FAD to Thr residues of flavoproteins in vitro and in vivo. Conclusion: ApbE is a novel modifying enzyme involved in the maturation of flavoproteins. Significance: Broad distribution of ApbE suggests a wide utilization of flavoproteins containing FMN attached via a phospho- ester bond. Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) contains two flavin residues as redox-active prosthetic groups attached by a phosphoester bond to threonine residues in subunits NqrB and NqrC. We demonstrate here that flavinylation of truncated Vibrio harveyi NqrC at Thr-229 in Escherichia coli cells requires the presence of a co-expressed Vibrio apbE gene. The apbE genes cluster with genes for Na+-NQR and other FMN-binding flavoproteins in bacterial genomes and encode proteins with previously unknown function. Experiments with isolated NqrC and ApbE proteins confirmed that ApbE is the only protein factor required for NqrC flavinylation and also indicated that the reaction is Mg2+-dependent and proceeds with FAD but not FMN. Inactivation of the apbE gene in Klebsiella pneumoniae, wherein the nqr operon and apbE are well separated in the chromosome, resulted in a complete loss of the quinone reductase activity of Na+-NQR, consistent with its dependence on covalently bound flavin. Our data thus identify ApbE as a novel modifying enzyme, flavin transferase.

Redox properties of the prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase. 2. Study of the enzyme by optical spectroscopy

Redox titration of the electronic spectra of the prosthetic groups of the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from Vibrio harveyi at different pH values showed five redox transitions corresponding to the four flavin cofactors of the enzyme and one additional transition reflecting oxidoreduction of the [2Fe-2S] cluster. The pH dependence of the measured midpoint redox potentials showed that the two-electron reduction of the FAD located in the NqrF subunit was coupled with the uptake of only one H(+). The one-electron reduction of neutral semiquinone of riboflavin and the formation of anion flavosemiquinone from the oxidized FMN bound to the NqrB subunit were not coupled to any proton uptake. The two sequential one-electron reductions of the FMN residue bound to the NqrC subunit showed pH-independent formation of anion radical in the first step and the formation of fully reduced flavin coupled to the uptake of one H(+) in the second step. All four flavins stayed in the anionic form in the fully reduced enzyme. None of the six redox transitions in Na(+)-NQR showed dependence of its midpoint redox potential on the concentration of sodium ions. A model of the sequence of electron transfer steps in the enzyme is suggested.

Redox properties of the prosthetic groups of Na(+)-translocating nadh:quinone oxidoreductase. 1. Electron paramagnetic resonance study of the enzyme

Redox properties of all EPR-detectable prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from Vibrio harveyi were studied at pH 7.5 using cryo-EPR spectroelectrochemistry. Titration shows five redox transitions. One with E(m) = -275 mV belongs to the reduction of the [2Fe-2S] cluster, and the four others reflect redox transitions of flavin cofactors. Two transitions (E(m)(1) = -190 mV and E(m)(2) = -275 mV) originate from the formation of FMN anion radical, covalently bound to the NqrC subunit, and its subsequent reduction. The remaining two transitions arise from the two other flavin cofactors. A high potential (E(m) = -10 mV) transition corresponds to the reduction of riboflavin neutral radical, which is stable at rather high redox potentials. An E(m) = -130 mV transition reflects the formation of FMN anion radical from a flavin covalently bound to the NqrB subunit, which stays as a radical down to very low potentials. Taking into account the EPR-silent, two-electron transition of noncovalently bound FAD located in the NqrF subunit, there are four flavins in Na(+)-NQR all together. Defined by dipole-dipole magnetic interaction measurements, the interspin distance between the [2Fe-2S](+) cluster and the NqrB subunit-bound FMN anion radical is found to be 22.5 +/- 1.5 A, which means that for the functional electron transfer between these two centers another cofactor, most likely FMN bound to the NqrC subunit, should be located.

Regulation of expression of Na+ -translocating NADH:quinone oxidoreductase genes in Vibrio harveyi and Klebsiella pneumoniae

The expression of genes encoding sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) was studied in the marine bacterium Vibrio harveyi and in the enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl concentration, pH value, and presence of an uncoupler in the growth media do not influence significantly the level of nqr expression. However, nqr expression depends on the growth substrates used by these bacteria. Na+-NQR is highly repressed in V. harveyi during anaerobic growth, and nqr expression is modulated by electron acceptors and values of their redox potentials. The latter effect was shown to be independent of the ArcAB regulatory system.

Primary steps of the Na+-translocating NADH:ubiquinone oxidoreductase catalytic cycle resolved by the ultrafast freeze-quench approach.

The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) is a component of respiratory chain of various bacteria, and it generates a redox-driven transmembrane electrochemical Na+ potential. Primary steps of the catalytic cycle of Na+-NQR from Vibrio harveyi were followed by the ultrafast freeze-quench approach in combination with conventional stopped-flow technique. The obtained sequence of events includes NADH binding (∼1.5 × 107 m–1 s–1), hydride ion transfer from NADH to FAD (∼3.5 × 103 s–1), and partial electron separation and formation of equivalent fractions of reduced 2Fe-2S cluster and neutral semiquinone of FAD (∼0.97 × 103 s–1). In the last step, a quasi-equilibrium is approached between the two states of FAD: two-electron reduced (50%) and one-electron reduced (the other 50%) species. The latter, neutral semiquinone of FAD, shares the second electron with the 2Fe-2S center. The transient midpoint redox potentials for the cofactors obtained during the fast kinetics measurements are very different from ones achieved during equilibrium redox titration and show that the functional states of the enzyme realized during its turning over cannot be modeled by the equilibrium approach.

Real-time kinetics of electrogenic Na(+) transport by rhodopsin from the marine flavobacterium Dokdonia sp. PRO95.

Discovery of the light-driven sodium-motive pump Na+-rhodopsin (NaR) has initiated studies of the molecular mechanism of this novel membrane-linked energy transducer. In this paper, we investigated the photocycle of NaR from the marine flavobacterium Dokdonia sp. PRO95 and identified electrogenic and Na+-dependent steps of this cycle. We found that the NaR photocycle is composed of at least four steps: NaR519 + hv → K585 → (L450↔M495) → O585 → NaR519. The third step is the only step that depends on the Na+ concentration inside right-side-out NaR-containing proteoliposomes, indicating that this step is coupled with Na+ binding to NaR. For steps 2, 3, and 4, the values of the rate constants are 4×104 s–1, 4.7 × 103 M–1 s–1, and 150 s–1, respectively. These steps contributed 15, 15, and 70% of the total membrane electric potential (Δψ ~ 200 mV) generated by a single turnover of NaR incorporated into liposomes and attached to phospholipid-impregnated collodion film. On the basis of these observations, a mechanism of light-driven Na+ pumping by NaR is suggested.