Nedielkov, Ruslan

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Crystallization and preliminary analysis of the NqrA and NqrC subunits of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae

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 NqrA1-377 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.


Localization of Ubiquinone-8 in the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

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.