2016, Vorburger, Thomas, Nedielkov, Ruslan, Brosig, Alexander, Bok, Eva, Schunke, Emina, Steffen, Wojtek, Mayer, Sonja, Götz, Friedrich, Möller, Heiko M., Steuber, Julia

For Vibrio cholerae, the coordinated import and export of Na⁺ is crucial for adaptation to habitats with different osmolarities. We investigated the Na⁺-extruding branch of the sodium cycle in this human pathogen by in vivo (23)Na-NMR spectroscopy. The Na⁺ extrusion activity of cells was monitored after adding glucose which stimulated respiration via the Na⁺-translocating NADH:quinone oxidoreductase (Na(+)-NQR). In a V. cholerae deletion mutant devoid of the Na⁺-NQR encoding genes (nqrA-F), rates of respiratory Na⁺ extrusion were decreased by a factor of four, but the cytoplasmic Na(+) concentration was essentially unchanged. Furthermore, the mutant was impaired in formation of transmembrane voltage (ΔΨ, inside negative) and did not grow under hypoosmotic conditions at pH8.2 or above. This growth defect could be complemented by transformation with the plasmid encoded nqr operon. In an alkaline environment, Na⁺/H⁺ antiporters acidify the cytoplasm at the expense of the transmembrane voltage. It is proposed that, at alkaline pH and limiting Na⁺ concentrations, the Na⁺-NQR is crucial for generation of a transmembrane voltage to drive the import of H(+) by electrogenic Na⁺/H⁺ antiporters. Our study provides the basis to understand the role of the Na⁺-NQR in pathogenicity of V. cholerae and other pathogens relying on this primary Na⁺ pump for respiration.

2013-08, Lavrynenko, Oksana, Nedielkov, Ruslan, Möller, Heiko M., Shevchenko, Andrej

Ecdysteroids are potent developmental regulators that control molting, reproduction, and stress response in arthropods. In developing larvae, picogram quantities of individual ecdysteroids and their conjugated forms are present along with milligrams of structural and energy storage lipids. To enhance the specificity and sensitivity of ecdysteroid detection, we targeted the 6-ketone group, which is common to all ecdysteroids, with Girard reagents. Unlike other ketosteroids, during the reaction, Girard hydrazones of ecdysteroids eliminated the C14-hydroxyl group, creating an additional C14-C15 double bond. Dehydrated hydrazones of endogenous ecdysteroids were detected by LC-MS/MS in the multiple reaction monitoring (MRM) mode using two mass transitions: one relied upon neutral loss of a quaternary amine from the Girard T moiety; another complementary transition followed neutral loss of the hydrocarbon chain upon C20-C27 cleavage. We further demonstrated that a combination of Girard derivatization and LC-MS/MS enabled unequivocal detection of three major endogenous hormones at the picogram level in an extract from a single Drosophila pupa.

2014, Vohl, Georg, Nedielkov, Ruslan, Claussen, Björn, Casutt, Marco S., Vorburger, Thomas, Diederichs, Kay, Möller, Heiko M., Steuber, Julia, Fritz, Günter

The Na⁺-translocating NADH:ubiquinone oxidoreductase (Na⁺-NQR) from Vibrio cholerae is a membrane protein complex consisting of six different subunits NqrA-NqrF. The major domains of the NqrA and NqrC subunits were heterologously expressed in Escherichia coli and crystallized. The structure of NqrA_1-377 was solved in space groups C222₁ and P2₁ by SAD phasing and molecular replacement at 1.9 and 2.1 Å resolution, respectively. NqrC devoid of the transmembrane helix was co-expressed with ApbE to insert the flavin mononucleotide group covalently attached to Thr225. The structure was determined by molecular replacement using apo-NqrC of Parabacteroides distasonis as search model at 1.8 Å resolution.

2011-11-18, Casutt, Marco S., Nedielkov, Ruslan, Wendelspiess, Severin, Vossler, Sara, Gerken, Uwe, Murai, Masatoshi, Miyoshi, Hideto, Möller, Heiko M., Steuber, Julia

Na + is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na+ -pumping NADH:quinone oxidoreductase (Na + -NQR) as the first complex in its respiratory chain. The Na + -NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na + translocation by the Na + -NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na + -NQR binds one molecule of ubiquinone-So Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-S bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na + -NQR is proposed that is initiated by NADH oxidation on sub unit NqrF and leads to quinol formation on subunit NqrA.

2013-10-18, Nedielkov, Ruslan, Steffen, Wojtek, Steuber, Julia, Möller, Heiko M.

The sodium ion-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na⁺-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc₁ and b₆f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na⁺-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na⁺-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na⁺-NQR.