Purification and characterization of the recombinant Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae.
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 P(BAD) 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 O(2) (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 Delta Psi 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.
Barquera
Blanca
B
Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Street, Urbana, Illinois 61801, USA. barquera@scs.uiuc.edu
Hellwig
Petra
P
Zhou
Weidong
W
Morgan
Joel E
JE
Häse
Claudia C
CC
Gosink
Khoosheh K
KK
Nilges
Mark
M
Bruesehoff
Peter J
PJ
Roth
Annette
A
Lancaster
C Roy D
CR
Gennis
Robert B
RB
D001483:Base Sequence; D016227:Benzoquinones / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D017136:Ion Transport; D009876:Operon; D016133:Polymerase Chain Reaction; D011808:Quinone Reductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D014734:Vibrio cholerae / Q000201:enzymology*
D016227:Benzoquinones; D017931:DNA Primers; D011994:Recombinant Proteins; C004532:quinone; D012964:Sodium; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases