Abstract
In the respiratory chain, NADH oxidation is coupled to ion translocation across the membrane to build up an electrochemical gradient. In the human pathogen Vibrio cholerae, the sodium-pumping NADH:quinone oxidoreductase (Na+-NQR) generates a sodium gradient by a so far unknown mechanism. Here we show that ion pumping in Na+-NQR is driven by large conformational changes coupling electron transfer to ion translocation. We have determined a series of cryo-EM and X-ray structures of the Na+-NQR that represent snapshots of the catalytic cycle. The six subunits NqrA, B, C, D, E, and F of Na+-NQR harbor a unique set of cofactors that shuttle the electrons from NADH twice across the membrane to quinone. The redox state of a unique intramembranous [2Fe-2S] cluster orchestrates the movements of subunit NqrC, which acts as an electron transfer switch. We propose that this switching movement controls the release of Na+ from a binding site localized in subunit NqrB.
![<div class='caption-title'>a, Close-up view of subunits NqrD-E coordinating a [2Fe-2S] cluster, with cysteines originating from both subunits. b, Localization of the [2Fe-2S] clusters in cytoplasmic NqrF and in membrane subunits NqrD-E. The blue circle indicates the [2Fe-2S]NqrF cluster; the red circle indicates the [2Fe-2S]NqrD-E cluster. c, CD spectra of wild-type (WT) Na+-NQR; NqrF-C70A, which is devoid of [2Fe-2S]NqrF; and NqrD-C29A, which is devoid of [2Fe-2S]NqrD-E. Spectra of WT Na+-NQR (black trace), the [2Fe-2S]NqrD-E cluster (red trace) in NqrF-C70A, and the [2Fe-2S]NqrF cluster (blue trace) in NqrD-C29A are shown. d, k3-weighted EXAFS and Fourier transform spectra of cluster [2Fe-2S]NqrD-E (red) in NqrF-C70A, and cluster [2Fe-2S]NqrF (blue) in NqrD-C29A. e, 57Fe Mössbauer spectrum of the [2Fe-2S]NqrD-E cluster in NqrF-C70A. f, 10 K X-band EPR spectrum of Na+-NQR variant NqrF-C70A showing [2Fe-2S]NqrD-E cluster, simulated with two components: a [2Fe-2S] cluster (orange; g||= 2.02, g = 1.94, line width = 50 G) and a radical signal (purple; giso = 2.01, line width = 20 G, 4% relative weight). g, Growth curves of V. cholerae expressing WT Na+-NQR (black) or Na+-NQR variants containing cluster [2Fe-2S]NqrD-E (red) in variant NqrF-C70A or cluster [2Fe-2S]NqrF (blue) in variant NqrD-C29A. OD600, optical density at 600 nm. Growth data are shown as mean ± s.d., n = 3 biologically independent samples.</div>](https://europepmc.org/articles/PMC10643135/bin/41594_2023_1099_Fig2_HTML.jpg "<div class='caption-title'>a, Close-up view of subunits NqrD-E coordinating a [2Fe-2S] cluster, with cysteines originating from both subunits. b, Localization of the [2Fe-2S] clusters in cytoplasmic NqrF and in membrane subunits NqrD-E. The blue circle indicates the [2Fe-2S]NqrF cluster; the red circle indicates the [2Fe-2S]NqrD-E cluster. c, CD spectra of wild-type (WT) Na+-NQR; NqrF-C70A, which is devoid of [2Fe-2S]NqrF; and NqrD-C29A, which is devoid of [2Fe-2S]NqrD-E. Spectra of WT Na+-NQR (black trace), the [2Fe-2S]NqrD-E cluster (red trace) in NqrF-C70A, and the [2Fe-2S]NqrF cluster (blue trace) in NqrD-C29A are shown. d, k3-weighted EXAFS and Fourier transform spectra of cluster [2Fe-2S]NqrD-E (red) in NqrF-C70A, and cluster [2Fe-2S]NqrF (blue) in NqrD-C29A. e, 57Fe Mössbauer spectrum of the [2Fe-2S]NqrD-E cluster in NqrF-C70A. f, 10 K X-band EPR spectrum of Na+-NQR variant NqrF-C70A showing [2Fe-2S]NqrD-E cluster, simulated with two components: a [2Fe-2S] cluster (orange; g||= 2.02, g = 1.94, line width = 50 G) and a radical signal (purple; giso = 2.01, line width = 20 G, 4% relative weight). g, Growth curves of V. cholerae expressing WT Na+-NQR (black) or Na+-NQR variants containing cluster [2Fe-2S]NqrD-E (red) in variant NqrF-C70A or cluster [2Fe-2S]NqrF (blue) in variant NqrD-C29A. OD600, optical density at 600 nm. Growth data are shown as mean ± s.d., n = 3 biologically independent samples.</div>")
PMID: 