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

Blanca Barquera, Petra Hellwig, Weidong Zhou, Joel E. Morgan, Claudia C. Häse, Khoosheh K. Gosink, Mark Nilges, Peter J. Bruesehoff, Annette Roth, C. Roy D. Lancaster, Robert B. Gennis

Research output: Contribution to journalArticlepeer-review

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 02 (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 δacross 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.

Original languageEnglish (US)
Pages (from-to)3781-3789
Number of pages9
JournalBiochemistry
Volume41
Issue number11
DOIs
StatePublished - Mar 19 2002

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Purification and characterization of the recombinant Na<sup>+</sup>-translocating NADH:quinone oxidoreductase from Vibrio cholerae'. Together they form a unique fingerprint.

Cite this