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.