Cryo-EM structures of Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae.

The Na+-pumping NADH-ubiquinone oxidoreductase (Na+-NQR) couples electron transfer from NADH to ubiquinone with Na+-pumping, generating an electrochemical Na+ gradient that is essential for energy-consuming reactions in bacteria. Since Na+-NQR is exclusively found in prokaryotes, it is a promising target for highly selective antibiotics. However, the molecular mechanism of inhibition is not well-understood for lack of the atomic structural information about an inhibitor-bound state. Here we present cryo-electron microscopy structures of Na+-NQR from Vibrio cholerae with or without a bound inhibitor at 2.5- to 3.1-Å resolution. The structures reveal the arrangement of all six redox cofactors including a herein identified 2Fe-2S cluster located between the NqrD and NqrE subunits. A large part of the hydrophilic NqrF is barely visible in the density map, suggesting a high degree of flexibility. This flexibility may be responsible to reducing the long distance between the 2Fe-2S centers in NqrF and NqrD/E. Two different types of specific inhibitors bind to the N-terminal region of NqrB, which is disordered in the absence of inhibitors. The present study provides a foundation for understanding the function of Na+-NQR and the binding manner of specific inhibitors.

The side chain of ubiquinone plays a critical role in Na+ translocation by the NADH-ubiquinone oxidoreductase (Na+-NQR) from Vibrio cholerae.

The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is an essential bacterial respiratory enzyme that generates a Na+ gradient across the cell membrane. However, the mechanism that couples the redox reactions to Na+ translocation remains unknown. To address this, we examined the relation between reduction of UQ and Na+ translocation using a series of synthetic UQs with Vibrio cholerae Na+-NQR reconstituted into liposomes. UQ0 that has no side chain and UQCH3 and UQC2H5, which have methyl and ethyl side chains, respectively, were catalytically reduced by Na+-NQR, but their reduction generated no membrane potential, indicating that the overall electron transfer and Na+ translocation are not coupled. While these UQs were partly reduced by electron leak from the cofactor(s) located upstream of riboflavin, this complete loss of Na+ translocation cannot be explained by the electron leak. Lengthening the UQ side chain to n-propyl (C3H7) or longer significantly restored Na+ translocation. It has been considered that Na+ translocation is completed when riboflavin, a terminal redox cofactor residing within the membrane, is reduced. In this view, the role of UQ is simply to accept electrons from the reduced riboflavin to regenerate the stable neutral riboflavin radical and reset the catalytic cycle. However, the present study revealed that the final UQ reduction via reduced riboflavin makes an important contribution to Na+ translocation through a critical role of its side chain. Based on the results, we discuss the critical role of the UQ side chain in Na+ translocation.

Specific chemical modification explores dynamic structure of the NqrB subunit in Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae.

The Na+-pumping NADH-ubiquinone oxidoreductase (Na+-NQR) is a main ion transporter in many pathogenic bacteria. We previously proposed that N-terminal stretch of the NqrB subunit plays an important role in regulating the ubiquinone reaction at the adjacent NqrA subunit in Vibrio cholerae Na+-NQR. However, since approximately three quarters of the stretch (NqrB-Met1-Pro37) was not modeled in an earlier crystallographic study, its structure and function remain unknown. If we can develop a method that enables pinpoint modification of this stretch by functional chemicals (such as spin probes), it could lead to new ways to investigate the unsettled issues. As the first step to this end, we undertook to specifically attach an alkyne group to a lysine located in the stretch via protein-ligand affinity-driven substitution using synthetic ligands NAS-K1 and NAS-K2. The alkyne, once attached, can serve as an "anchor" for connecting functional chemicals via convenient click chemistry. After a short incubation of isolated Na+-NQR with these ligands, alkyne was predominantly incorporated into NqrB. Proteomic analyses in combination with mutagenesis of predicted target lysines revealed that alkyne attaches to NqrB-Lys22 located at the nonmodeled region of the stretch. This study not only achieved the specific modification initially aimed for but also provided valuable information about positioning of the nonmodeled region. For example, the fact that hydrophobic NAS-Ks come into contact with NqrB-Lys22 suggests that the nonmodeled region may orient toward the membrane phase rather than protruding into cytoplasmic medium. This conformation may be essential for regulating the ubiquinone reaction in the adjacent NqrA.