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  Purification and characterization of the recombinant Na+-translocating NADH : quinone oxidoreductase from vibrio cholerae

Barquera, B., Hellwig, P., Zhou, W., Morgan, J. E., Häse, C. C., Gosink, K. K., et al. (2002). Purification and characterization of the recombinant Na+-translocating NADH: quinone oxidoreductase from vibrio cholerae. Biochemistry, 41(11), 3781-3789. doi:10.1021/bi011873o.

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Barquera, Blanca1, Author
Hellwig, Petra2, Author
Zhou, Weidong1, Author
Morgan, Joel E.1, Author
Häse, Claudia C.3, Author
Gosink, Khoosheh K3, Author
Nilges, Mark4, Author
Bruesehoff, Peter J.5, Author
Roth, Annette6, Author           
Lancaster, C. Roy D.6, Author           
Gennis, Robert B.1, Author
1Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA, ou_persistent22              
2Institut für Biophysik Universität Frankfurt, 60590 Frankfurt am Main, Germany, ou_persistent22              
3Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105-2794, USA, ou_persistent22              
4Illinois EPR Research Center, Urbana, Illinois 61801, USA, ou_persistent22              
5Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA, ou_persistent22              
6Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              


Free keywords: Coli dna photolyase; Ubiquinone oxidoreductase; Escherichia-coli; Sodium cycle; Alginolyticus; Reductase; Marine; Flavin; Bacteria; Ag+
 Abstract: The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), was cloned under the regulation of the PBAD promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics studies of this sodium-pumping enzyme, a host strain of V. cholerae was constructed in which the genomic copy of the nqr operon was deleted. By using a vector containing a six-histidine tag on the carboxy terminus of the NqrF subunit, the last subunit in the operon, the recombinant enzyme was readily purified by affinity chromatography in a highly active form from detergent-solubilized membranes of V. cholerae. The recombinant enzyme has a high specific activity in the presence of sodium. NADH consumption was assessed at a turnover number of 720 electrons per second. When purified using dodecyl maltoside (DM), the isolated enzyme contains approximately one bound ubiquinone, whereas if the detergent LDAO is used instead, the quinone content of the isolated enzyme is negligible. Furthermore, the recombinant enzyme, purified with DM, has a relatively low rate of reaction with O2 (10-20 s(-1)). In steady state turnover, the isolated, recombinant enzyme exhibits up to 5-fold stimulation by sodium and functions as a primary sodium pump, as reported previously for Na+-NQR from other bacterial sources. When reconstituted into liposomes, the recombinant Na+-NQR generates a sodium gradient and a Deltapsi across the membrane. SDS-PAGE resolves all six subunits, two of which, NqrB and NqrC, contain covalently bound flavin. A redox titration of the enzyme, monitored by UV-visible spectroscopy, reveals three n = 2 redox centers and one n = 1 redox center, for which the presence of three flavins and a 2Fe-2S center can account. The V. cholerae Na+-NQR is well-suited for structural studies and for the use of molecular genetics techniques in addressing the mechanism by which NADH oxidation is coupled to the pumping of Na+ across the membrane.


Language(s): eng - English
 Dates: 2001-10-022002-01-282002-02-232002-03-19
 Publication Status: Published in print
 Pages: 9
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/bi011873o
 Degree: -



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Title: Biochemistry
Source Genre: Journal
Publ. Info: Columbus, Ohio : American Chemical Society
Pages: - Volume / Issue: 41 (11) Sequence Number: - Start / End Page: 3781 - 3789 Identifier: ISSN: 0006-2960
CoNE: https://pure.mpg.de/cone/journals/resource/954925384103