Strong pH dependence of coupling efficiency of the Na⁺ – translocating NADH:quinone oxidoreductase (Na⁺-NQR) of Vibrio cholerae

Abstract

The Na+-translocating NADH:quinone oxidoreductase (NQR) is the entry site for electrons into the respiratory chain of Vibrio cholerae, the causative agent of cholera disease. NQR couples the electron transfer from NADH to ubiquinone to the translocation of sodium ions across the membrane. We investigated the pH dependence of electron transfer and generation of a transmembrane voltage (ΔΨ) by NQR reconstituted in liposomes with Na+ or Li+ as coupling cation. ΔΨ formation was followed with the voltage-sensitive dye oxonol. With Na+, ΔΨ was barely influenced by pH (6.5-8.5), while Q reduction activity exhibited a maximum at pH 7.5-8.0. With Li+, ΔΨ was generally lower, and the pH profile of electron transfer activity did not reveal a pronounced maximum. We conclude that the coupling efficiency of NQR is influenced by the nature of the transported cation, and by the concentration of protons. The 3D structure of NQR reveals a transmembrane channel in subunit NqrB. It is proposed that partial uncoupling of the NQR observed with the smaller Li+, or with Na+ at pH 7.5-8.0, is caused by the backflow of the coupling cation through the channel in NqrB.

Identification of the riboflavin cofactor-binding site in the Vibrio cholerae ion-pumping NQR complex: A novel structural motif in redox enzymes

“…, FAD and FMN), riboflavin goes more deeply in the proposed pocket ( Fig. 3 A ), which is capped by subunit A that sits on top, consistent with the pH titration data, showing that the riboflavin cofactor is unable to exchange protons with the aqueous environment ( 40 ). Moreover, a buried site is also consistent with the environment required to shield the riboflavin neutral radical from oxygen and other strong oxidants ( 23 , 32 , 41 ).…”

Occurrence and Function of the Na+-Translocating NADH:Quinone Oxidoreductase in Prevotella spp.

“…NADH oxidation activity of P. bryantii B 1 4 membranes increased from 223 nmol min −1 mg −1 at a NaCl concentration of 100 µM to a maximum of 334 nmol min −1 mg −1 at a NaCl concentration of 500 µM. Please note that the residual Na + concentration in assay buffers (40 µM) would be sufficient for half-maximal activity of purified NQR from Vibrio cholera [52], which explains the basal activity of P. bryantii B 1 4 membranes without added salts (Figure 5). The inhibition of NADH oxidation activity by silver ions [49,53], and the stimulation of NADH oxidation by Na + [54] are hallmark features for the presence of NQR in bacterial membranes, and demonstrate that P. bryantii B 1 4 membranes also contain active NQR.…”