title: The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae enhances insertion of FeS in overproduced NqrF subunit.
abstract: The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae is a membrane-bound, respiratory Na+ pump. Its NqrF subunit contains one FAD and a [2Fe-2S] cluster and catalyzes the initial oxidation of NADH. A soluble variant of NqrF lacking its hydrophobic, N-terminal helix (NqrF') was produced in V. cholerae wild type and nqr deletion strain. Under identical conditions of growth and induction, the yield of NqrF' increased by 30% in the presence of the Na+-NQR. FAD-containing NqrF' species with or without the FeS cluster were observed, indicating that assembly of the FeS center, but not insertion of the flavin cofactor, was limited during overproduction in V. cholerae. A comparison of these distinct NqrF' species with regard to specific NADH dehydrogenase activity, pH dependence of activity and thermal inactivation showed that NqrF' lacking the [2Fe-2S] cluster was less stable, partially unfolded, and therefore prone to proteolytic degradation in V. cholerae. We conclude that the overall yield of NqrF' critically depends on the amount of fully assembled, FeS-containing NqrF' in the V. cholerae host cells. The Na+-NQR is proposed to increase the stability of NqrF' by stimulating the maturation of FeS centers.
authors.affiliation: Biochemisches Institut, Universität Zürich, CH-8057 Zürich, Switzerland.
authors.forename: Minli
authors.initials: M
authors.lastname: Tao
authors.forename: Günter
authors.initials: G
authors.lastname: Fritz
authors.forename: Julia
authors.initials: J
authors.lastname: Steuber
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D005420:Flavoproteins / Q000096:biosynthesis / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D011808:Quinone Reductases / Q000302:isolation & purification / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D014734:Vibrio cholerae / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D005420:Flavoproteins; D007506:Iron-Sulfur Proteins; D021122:Protein Subunits; D009247:NADH, NADPH Oxidoreductases; C099916:Nqrf protein, Vibrio alginolyticus; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases