Edited by Ruma BanerjeeThe Na ؉ -pumping NADH-quinone oxidoreductase (Na ؉ -NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae. I]PAD-2, rather than being competitively suppressed in the presence of other inhibitors, is enhanced under some experimental conditions. To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na ؉ -NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work.The Na ϩ -pumping NADH-quinone oxidoreductase (Na ϩ -NQR) 2 is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, such as Vibrio cholerae and Haemophilus influenzae (1, 2). Na ϩ -NQR obtains energy by oxidizing NADH and reducing ubiquinone, which allows it to generate an electrochemical Na ϩ gradient across the inner bacterial membrane. The enzyme is an integral membrane complex consisting of six subunits (NqrA-F) encoded by the nqr operon (2). There is a consensus that the electron transfer takes place through a series of five redox cofactors as follows: a FAD; a 2Fe-2S center; two covalently bound FMN, and a riboflavin (3-5). Different studies have intensively investigated the locations and redox properties of the cofactors of the enzyme (6 -12); however, the exact locations of the ubiquinone-binding site(s) and the Na ϩ transport pathway, as well as the mechanism that couples Na ϩ transport to the electron transfer, still remain elusive.A recently published X-ray crystallographic study provided important information about the structure of V. cholerae Na ϩ -NQR (13), but the model includes several features that are difficult to reconcile with previous biochemical and/or biophysical functional characterizations (11). For instance, the spatial distances between several pairs of redox cofactors in the proposed electron transfer pathway are too large to support physiological rates of electron transfer; for example, the edge-toedge distance between the [2Fe-2S] cluster in NqrF and the Fe(Cys) 4 in NqrD (33.4 Å) and between the FMN and the riboflavin cofactor in NqrB (29.3 Å). The fact that electron transfer between these cofactors takes place indicates that the subunits harboring the cofactors undergo large conformational changes during turnover that decrease these spatial gaps (13).Additionally, the crystallographic model lacks an anticipated tightly bound quinone, which has been reported in the enzyme preparations from different laboratories (4,6,8) and suggested to be located in NqrA on the basis of photoaffinity labeling and NMR studies (7,9). In the crystallographic model, the NqrA subunit includes a deep cavity that is large enough to accommodate a ubiquinone molecule, but the cavity is ϳ20 Å above the predicted membrane surface and the distance between the cavity and the riboflavin in NqrB subunit is too large (Ͼ40 Å) to be consistent with electron transfer during tu...