Conformational coupling of redox-driven Na+-translocation in Vibrio cholerae NADH:quinone oxidoreductase

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.


Movement of ferredoxin-like domain of NqrF
In the different cryo-EM and X-ray structures reported here, the ferredoxin-like domain of NqrF resides in different positions (Supplementary Fig. 1) illustrating that this domain is rather flexibly linked between the transmembrane helix and the FNR-like domain.E.g., aligning the NqrF subunit on the transmembrane helix, the Ca positions of the ferredoxin-like domain in the X-ray structure versus the cryo-EM structure with NADH and Q2 differ by a distance of 4-8 Å Angstrom.The position of the [2Fe-2S] cluster differs in these structures by 4.5 Å (Supplementary Fig. 1).Thus, the ferredoxin-like domain is flexibly tethered between the FAD-binding domain and the transmembrane helix allowing for large translational freedom, whereas the FNR-like domain interacts with NqrA that serves as a pivot point for rotational movements.This arrangement is key for domain motion of NqrF upon binding of NADH.The cryo-EM structure of Na + -NQR reacted with NADH at 2.55 Å resolution shows that the ferredoxin-like domain of NqrF harbouring a [2Fe-2S] cluster approaches the membrane in a tilting motion, while the FNR-like domain of NqrF slides sideways to make space for the ferredoxin-like domain (Supplementary Video 1, Supplementary Fig. 1).NqrD-E (NqrD-C29A).These variants show only half of the Fe or acid labile sulphide content compared to wildtype Na + -NQR (Supplementary Table 1) and largely reduced quinone reduction activity (Supplementary Table 2).Wildtype and variants of Na + -NQR were analysed by a complementary set of several spectroscopic techniques including circular dichroism (CD), EPR, 57 Fe Mössbauer, and Fe Ka high-energy fluorescence detected (HERFD) X-ray absorption spectroscopy (XAS).

Supplementary Table 1. Detection of iron and acid-labile sulphide in wt Na + -NQR and variants
The amount of iron and acid-labile sulphide was determined in Na + -NQR and its variants.The amount of Fe found with inductively coupled plasma resonance mass spectroscopy was correlated to the sulphur content in the same sample.Acid-labile sulphide was determined colorimetrically.Mean values from three technical replicates of three independent protein preparations (n = 9) and standard deviations are given.APS reductase from Desulfovibrio desulfuricans served and bovine serum albumin served as controls.

NQR variants
Mol Fe : mol protein Mol S

Supplementary Table 2. Electron transfer activity of wt Na + -NQR and variants
Sodium dependent oxidation of NADH and reduction of ubiquinone-1 by Na + -NQR was determined spectrophotometrically at 340 nm or 282 nm, respectively.The reaction was started with the addition of Na + -NQR in the presence of saturating concentrations of ubiquinone-1 (UQ-1) (0.1 mM) and NADH (0.15 mM).The residual Na + concentration in the assay buffer was 20 µM Na + .Standard derivations and mean of n= independent 3 experiments are given.

HERFD-XAS Fe K-edge and analysis of the [2Fe-2S] clusters in NqrF and NqrD-E
The Fe K-pre-edge XAS at 7112 eV of the NqrD-C70A sample was slightly shifted to higher energy indicating a distortion of the geometry of the [2Fe-2S]NqrD-E cluster (Supplementary Fig. 2a).Further evidence for such a distorted geometry comes also from the low field of X-band EPR spectra (see below and Supplementary Data Fig. 2 b-d).The EXAFS of both NqrF-C70A and NqrD-C29A exhibit an intense scattering shell in the non-phase shift corrected Fourier transform of the EXAFS at a radial distance of 1.90 Å that corresponds to degenerate Fe-S scattering interactions, (Supplementary Fig. 2a).This interaction is generally well-fit with a four-fold degenerate Fe-S scattering path at mean scattering distance of 2.28 Å (Fits 1 and 5, Supplementary Table 3, Supplementary Fig. 3).Furthermore, a second less intense scattering shell is observed at a longer radial distance of 2.40 Å that corresponds to an Fe-Fe scattering interaction.Inclusion of a single Fe-Fe scattering interaction at 2.7 Å significantly improves the EXAFS fits of both NqrF-C70A and NqrD-C29A (Fits 2 and 6, respectively), as evidenced by a significant reduction of the χ 2 value.These EXAFS fits clearly assign the iron cofactors of NqrF-C70A and NqrD-C29A as [2Fe-2S] clusters.

EPR spectroscopic analysis of the [2Fe-2S] clusters in NqrF and NqrD-E
The EPR spectra of the dithionite reduced [2Fe-2S] cluster in NqrF exhibits features resembling vertebrate-type ferredoxins and has been described by us in previous studies 1,2 (Supplementary Fig. 2b).EPR spectra of the so far uncharacterized intramembranous cluster in NqrD-E exhibited a weak signal with two distinct features (Supplementary Fig. 2b,e).The prominent feature at g ~ 2.01 is consistent with a microwave power saturated radical signal, while the feature at g ~ 1.94 was only observed under relatively high microwave powers, indicative for a fast-relaxing species.The assigned g-values of the NqrD-E cluster are similar to that observed for the NqrF cluster 23 , but we do note the potentially very different relaxation behaviours of the two clusters as indicated by differences in microwave power required for observation.The high-field feature (Supplementary Fig. 2c,d) resembles the g⊥ of various [2Fe-2S] clusters 21,22 , however, a distinct corresponding g|| feature of an axial EPR spectrum in the expected range of 2.01 to 2.06 is not observed.At low field an additional feature at g=6.4 was observed indicating the presence of some cluster in a higher spin state than S=1/2 (Supplementary Fig. 2 c,d).The feature is reminiscent of a [2Fe-2S] cluster with an non-Cys ligand in the coordination sphere that exhibits a S=9/2 giving rise to a EPR transition with a feature at g=6. and Supplementary Data Fig. 5).
Reaction of wt-NQR with NADH at 20° C results in rapid reduction of the FAD in NqrF subunit already within the dead time of the instrument.Subsequently, electrons are transferred from FAD to the other cofactors (Supplementary Fig. 5a).The first electron steps can be described by first order kinetics and were fitted by linear regression in semi-logarithmic plots.Reduction of the [2Fe-2S] cluster in NqrF within 5 milliseconds was observed.Clearly, also the intramembranous [2Fe-2S]NqrD-E cluster was reduced (Supplementary Fig. 5a) during course of the reaction, which was confirmed by a decrease of the absorption at 575 nm (Supplementary Fig. 5c).A third rate observed represents the reduction of the FMN in NqrC.
In contrast the reaction of variant NqrD-C29A with NADH resulted only in partial reduction of the cofactors (Supplementary Fig. 5b).No further spectral change was observed even after

Cryo-EM map sharpening and density modifications
Different map sharpening procedures were applied throughout the entire process of model building and the outcome of the different sharpening procedures have been extensively compared to yield the best possible maps.Best results with respect to recognisable details were obtained by density modifications and phenix.resolve_cryo-em 4 and resampling of the final map at 0.3 -0.5 Å/pixel using relion_image_handler 5 (Supplementary Fig. 7 a-c).We have applied regularly phenix.auto_sharpen 6, LocalDeBlur 7 , LocScale 8 or DeepEMhancer 9 .
phenix.auto_sharpen gave often satisfying results, while e.g.DeepEMhancer yielded maps with better connectivity, in particular for map regions which have been weak due to flexibility of the domains, like e.g.NqrF (Supplementary Fig. 6i-m).

Figure 1 .b
Structural snapshots of rotational flexibility of the ferredoxin-like domain of NqrF.The ferredoxin-like domain of NqrF resides at different positions in the different cryo-EM and X-ray structures.a, Left: Structural alignment of the NqrF ferredoxin domain and the transmembrane helix domain.The FAD containing FNR-like domain is omitted for clarity.There is a rotational and a transversal movement of the ferredoxin domain relative to the transmembrane helix.For clarity, only the X-ray structure (orange), the cryo-EM structure with inhibitor HQNO (red) and the X-ray structure with inhibitor DQA (dark red) are shown.Right: extrapolation (grey) of the movement of the ferredoxin-like domain towards the membrane plane.b, Different conformations of the NqrF ferredoxin domain observed in this study with respect to the transmembrane helix.Broken lines indicate most distant and most proximate position of ferredoxin domain from the membrane, and most distant and proximate position of the [2Fe-2S] cluster from the membrane.a Analysis of the intramembranous [2Fe-2S] cluster in NqrD-E In order to study the [2Fe-2S] clusters in NqrF or in NqrD-E individually, we have generated variants that lack either the [2Fe-2S] cluster in NqrF (NqrF-C70A) or the [2Fe-2S] cluster in

1
second.The variant lacking [2Fe-2S] in NqrD-E showed like wt Na + -NQR rapid reduction of FAD and subsequent electron transfer to the [2Fe-2S] located in NqrF.However, electron transfer in NqrD-C29A lacking [2Fe-2S]NqrD-E was interrupted at NqrF and no further reduction of FMN in NqrC was observed.No change in absorption was absorbed at 575 nm, in agreement with the absence of cluster [2Fe-2S]NqrD-E (Supplementary Fig. 4c).