The Electron Transfer Pathway of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

Abstract

The Na ؉ -pumping NADH:quinone oxidoreductase (Na ؉ -NQR) is the only respiratory enzyme that operates as a Na ؉ pump. This redox-driven Na ؉ pump is amenable to experimental approaches not available for H ؉ pumps, providing an excellent system for mechanistic studies of ion translocation. An understanding of the internal electron transfer steps and their Na ؉ dependence is an essential prerequisite for such studies. To this end, we analyzed the reduction kinetics of the wild type Na ؉ -NQR, as well as site-directed mutants of the enzyme, which lack specific cofactors. NADH and ubiquinol were used as reductants in separate experiments, and a full spectrum UVvisible stopped flow kinetic method was employed. The results make it possible to define the complete sequence of redox carriers in the electrons transfer pathway through the enzyme. Electrons flow from NADH to quinone through the FAD in subunit F, the 2Fe-2S center, the FMN in subunit C, the FMN in subunit B, and finally riboflavin. The reduction of the FMN C to its anionic flavosemiquinone state is the first Na ؉ -dependent process, suggesting that reduction of this site is linked to Na The Na ϩ -pumping NADH: ubiquinone oxidoreductase (Na ϩ -NQR) 2 is a molecular energy transducer present in the membrane of many marine and pathogenic bacteria, including Vibrio cholerae (1). The enzyme accepts electrons from NADH and donates them to ubiquinone. This redox reaction releases a significant amount of energy, which is used to pump Na ϩ across the cell membrane, creating a Na ϩ gradient as well as an electrical potential (2, 3). Na ϩ -NQR catalyzes the same redox reaction as the H ϩ -pumping NADH:quinone oxidoreductase (complex I), but there is nothing to indicate a common mechanism. The two proteins have significantly different cofactor compositions and do not share any homology beyond substrate/cofactor motifs. In fact the pumping stoichiometry of the two enzymes is reportedly different; Na ϩ -NQR translocates one Na ϩ /electron, whereas as complex I moves at least 2 H ϩ /e Ϫ (4 -6).In Na ϩ -NQR electrons are carried through the enzyme by means of at least five redox cofactors: a noncovalently bound FAD and a 2Fe-2S center, in the NqrF subunit, where the binding site for NADH is also located, two covalently attached FMNs in subunits B and C (FMN B and FMN C , respectively), and a noncovalently bound riboflavin that has not been definitely localized but that is believed to reside in the B or C subunit (see Fig. 1) (7-12). Describing the way in which these redox cofactors work together to move electrons from NADH to quinone is a necessary step in understanding how Na ϩ -NQR functions as a molecular energy converter. It is important to know the sequence of redox carriers along the path taken by electrons as they flow through the enzyme, as well as the kinetics and energetics of each step and how they are influenced by Na ϩ and the electrical potential.The reaction of the oxidized enzyme with excess NADH, leading to fully reduced state, can be used to investigate t...

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

“…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.…”

“…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.…”

“…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).…”

“…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)(4)(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.…”

Energy transducing redox steps of the Na + -pumping NADH:quinone oxidoreductase from Vibrio cholerae

“…Studies carried out by our group with mutants that do not incorporate individual cofactors have shown that electrons move through the enzyme following the pathway: FAD → 2Fe-2S → FMN C → FMN B → Riboflavin ( Fig. 1) (14).…”