NMR Reveals Double Occupancy of Quinone-type Ligands in the Catalytic Quinone Binding Site of the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae

Abstract

Background:The Na ϩ -NQR is a respiratory Na ϩ pump found in prokaryotes.

The structure of Na+-translocating of NADH:ubiquinone oxidoreductase of Vibrio cholerae: implications on coupling between electron transfer and Na+ transport

“…With Na + -NQR solubilized in lauryl-dimethyl-amine N-oxide, Q8 was lost from the enzyme Casutt et al, 2011). We recently showed that the quinone-binding site in NqrA could accommodate two quinone molecules in close proximity (Nedielkov et al, 2013). With eight isoprenoid units, Q8 is a very hydrophobic molecule, but its redox-reactive quinone head group is rather hydrophilic.…”

Identification of the binding sites for ubiquinone and inhibitors in the Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae by photoaffinity labeling

“…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 turnover (Fig.…”

“…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 turnover (Fig.…”

“…As specific inhibitors of many respiratory enzymes disturb the enzyme function, in general, by interfering with the redox reaction of quinone/quinol, identification of the binding site for inhibitors is also invaluable. The inhibitors that have been used for previous Na ϩ -NQR research are limited to a few commercially available chemicals such as HQNO (8,9), which has only moderate inhibitory potency (IC 50 or K i value in the micromolar range). Korormicin (Fig.…”

The Na + -Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae

“…Cells were harvested, washed once with 50 mM sodium phosphate (pH 8.0) and 0.5 M NaCl and stored at Ϫ80°C. Disruption of cells in a continuous lysis system (Emulsiflex C3 homogenizer; Avestin) and preparation of membranes were performed as described previously (28). The membranes were suspended in 50 mM sodium phosphate (pH 8.0) containing 0.3 M NaCl and 5% (wt/vol) glycerol to a final concentration of approximately 10 mg protein ml Ϫ1 as determined by the bicinchoninic acid assay (29).…”