Purification and Characterization of the Recombinant Na⁺-Translocating NADH:Quinone Oxidoreductase from Vibrio cholerae

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 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.

Riboflavin is a component of the Na + -pumping NADH–quinone oxidoreductase from Vibrio cholerae

“…Recombinant Na ϩ -NQR from V. cholerae was purified according to a protocol described before (10). The V. harveyi Na ϩ -NQR was prepared as described (9).…”

“…Na ϩ -NQR is the entry point for electrons into the respiratory chain of a number of marine and pathogenic bacteria (5-7). The enzyme has been isolated from several of these bacteria (8,9) including Vibrio cholerae (10). The nqr operon from V. cholerae has been cloned and expressed in the parent organism (10), and the purified recombinant enzyme has been used in the current studies.…”

“…The purified Na ϩ -NQR is an assembly of six polypeptide subunits (NqrA-F; ref. 10). Analysis of the polypeptide sequences indicates that five of the subunits contain transmembrane helices (11).…”

“…It is probable that the FAD and the [2Fe-2S] center are the initial electron acceptors from NADH. The covalently bound FMNs are attached to NqrB and NqrC by phosphodiester linkages through threonine residues (10,13). This mode of covalent flavin binding is unique to Na ϩ -NQR.…”

“…This mode of covalent flavin binding is unique to Na ϩ -NQR. In addition, Na ϩ -NQR contains an EPR-detectable radical that is observed in both the airoxidized and dithionite-reduced forms of the enzyme (8,10). EPR and electron nuclear double resonance spectroscopies have unequivocally established that the radical is present in a stoichiometry of 1 equivalent per mol of enzyme and is a neutral flavin-semiquinone in the oxidized form of the enzyme and an anionic flavin semiquinone in the NADH or dithionite-reduced forms of Na ϩ -NQR.…”