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

Abstract

SUMMARYThe 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 riboflavin, whose position has been under debate, and a newly assigned 2Fe-2SNqrD/E cluster located between the membrane embedded NqrD and NqrE subunits. A large part of the hydrophilic NqrF near the cytoplasmic membrane surface 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, consistent with physiologically relevant electron transfer. Two different types of specific inhibitors (korormicin A and aurachin D-42) bind to the N-terminal region of NqrB, which is disordered in the absence of inhibitors. The current inhibitor-bound structures reasonably explain our previous biochemical findings obtained by different chemistry-based experiments. This study provides a definite foundation for understanding the function of Na+-NQR and the molecular mechanism of its specific inhibitors to support molecular design of new antibiotics targeting the enzyme.

Identification of complex III, NQR, and SDH as primary bioenergetic enzymes during the stationary phase of Pseudomonas aeruginosa cultured in urine-like conditions

“…While complex I appears to be more energy efficient, there are several advantages of NQR that could explain why P. aeruginosa uses it as the main NADH dehydrogenase. For instance, complex I contains two 2Fe-2S and six 4Fe-2S clusters (Yagi, 1991;Yagi et al, 1998;Kerscher et al, 2008), while NQR contains only two 2Fe-2S centers (Kishikawa et al, 2022).…”