Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.
Advertisement
Nature (2022)
3192
147
Metrics details
Emissions of the critical ozone-depleting and greenhouse gas nitrous oxide (N2O) from soils and industrial processes have increased considerably over the last decades1,2,3. As the final step of bacterial denitrification, N2O is reduced to chemically inert N2 (refs. 1,4) in a reaction that is catalysed by the copper-dependent nitrous oxide reductase (N2OR) (ref. 5). The assembly of its unique [4Cu:2S] active site cluster CuZ requires both the ATP-binding-cassette (ABC) complex NosDFY and the membrane-anchored copper chaperone NosL (refs. 4,6). Here we report cryo-electron microscopy structures of Pseudomonas stutzeri NosDFY and its complexes with NosL and N2OR, respectively. We find that the periplasmic NosD protein contains a binding site for a Cu+ ion and interacts specifically with NosL in its nucleotide-free state, whereas its binding to N2OR requires a conformational change that is triggered by ATP binding. Mutually exclusive structures of NosDFY in complex with NosL and with N2OR reveal a sequential metal-trafficking and assembly pathway for a highly complex copper site. Within this pathway, NosDFY acts as a mechanical energy transducer rather than as a transporter. It links ATP hydrolysis in the cytoplasm to a conformational transition of the NosD subunit in the periplasm, which is required for NosDFY to switch its interaction partner so that copper ions are handed over from the chaperone NosL to the enzyme N2OR.
Your institute does not have access to this article
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
$29.99
monthly
Subscribe to Journal
Get full journal access for 1 year
$199.00
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Buy article
Get time limited or full article access on ReadCube.
$32.00
All prices are NET prices.
The atomic coordinates of the NosDFY, NosFY and NosDFY–AMPPNP in GDN as well as NosDFY and NosDF(E154Q)Y–ATP, NosDFYL and NosZDF(E154Q)Y–ATP in DDM micelles have been deposited with the PDB at http://www.pdb.org. Several models were uploaded for the different states of the R domain. The three-dimensional cryo-EM reconstructions of the masked maps post-processing have been deposited with the Electron Microscopy Data Bank (EMDB). Accession numbers of the individual models in the PDB and EMDB are listed in Extended Data Table 1.
Thompson, R. L. et al. Acceleration of global N2O emissions seen from two decades of atmospheric inversion. Nat. Clim. Chang. 9, 993–997 (2019).
ADS CAS Article Google Scholar
Ravishankara, A. R., Daniel, J. S. & Portmann, R. W. Nitrous oxide (N2O): the dominant ozone-depleting substance emitted in the 21st century. Science 326, 123–125 (2009).
ADS CAS PubMed Article Google Scholar
Tian, H. Q. et al. A comprehensive quantification of global nitrous oxide sources and sinks. Nature 586, 248–256 (2020).
ADS CAS PubMed Article Google Scholar
Honisch, U. & Zumft, W. G. Operon structure and regulation of the nos gene region of Pseudomonas stutzeri, encoding an ABC-type ATPase for maturation of nitrous oxide reductase. J. Bacteriol. 185, 1895–1902 (2003).
CAS PubMed PubMed Central Article Google Scholar
Zumft, W. G. & Kroneck, P. M. H. Respiratory transformation of nitrous oxide (N2O) to dinitrogen by Bacteria and Archaea. Adv. Microb. Physiol. 52, 107–225 (2007).
CAS PubMed Article Google Scholar
Pomowski, A., Zumft, W. G., Kroneck, P. M. H. & Einsle, O. N2O binding at a [4Cu:2S] copper–sulphur cluster in nitrous oxide reductase. Nature 477, 234–237 (2011).
ADS CAS PubMed Article Google Scholar
Thomson, A. J., Giannopoulos, G., Pretty, J., Baggs, E. M. & Richardson, D. J. Biological sources and sinks of nitrous oxide and strategies to mitigate emissions. Phil. Trans. R. Soc. B 367, 1157–1168 (2012).
CAS PubMed PubMed Central Article Google Scholar
Butterbach-Bahl, K., Baggs, E. M., Dannenmann, M., Kiese, R. & Zechmeister-Boltenstern, S. Nitrous oxide emissions from soils: how well do we understand the processes and their controls? Phil. Trans. R. Soc. B 368, 20130122 (2013).
PubMed PubMed Central Article CAS Google Scholar
Jansson, J. K. & Hofmockel, K. S. Soil microbiomes and climate change. Nat. Rev. Microbiol. 18, 35–46 (2020).
CAS PubMed Article Google Scholar
Stein, L. Y. The long-term relationship between microbial metabolism and greenhouse gases. Trends Microbiol. 28, 500–511 (2020).
CAS PubMed Article Google Scholar
Torres, M. J. et al. Nitrous oxide metabolism in nitrate-reducing bacteria: physiology and regulatory mechanisms. Adv. Microb. Physiol. 68, 353–432 (2016).
CAS PubMed Article Google Scholar
Pauleta, S. R., Carepo, M. S. P. & Moura, I. Source and reduction of nitrous oxide. Coord. Chem. Rev. 387, 436–449 (2019).
CAS Article Google Scholar
Solomon, E. I. et al. Copper active sites in biology. Chem. Rev. 114, 3659–3853 (2014).
CAS PubMed PubMed Central Article Google Scholar
Dupont, C. L., Grass, G. & Rensing, C. Copper toxicity and the origin of bacterial resistance—new insights and applications. Metallomics 3, 1109–1118 (2011).
CAS PubMed Article Google Scholar
Schneider, L. K. & Einsle, O. Role of calcium in secondary structure stabilization during maturation of nitrous oxide reductase. Biochemistry 55, 1433–1440 (2016).
CAS PubMed Article Google Scholar
Locher, K. P. Mechanistic diversity in ATP-binding cassette (ABC) transporters. Nat. Struct. Mol. Biol. 23, 487–493 (2016).
CAS PubMed Article Google Scholar
Higgins, C. F. ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol. 8, 67–113 (1992).
CAS PubMed Article Google Scholar
Li, Y. Y., Orlando, B. J. & Liao, M. F. Structural basis of lipopolysaccharide extraction by the LptB2FGC complex. Nature 567, 486–490 (2019).
ADS CAS PubMed PubMed Central Article Google Scholar
Fitzpatrick, A. W. P. et al. Structure of the MacAB–TolC ABC-type tripartite multidrug efflux pump. Nat. Microbiol. 2, 17070 (2017).
CAS PubMed PubMed Central Article Google Scholar
Zumft, W. G., Viebrock-Sambale, A. & Braun, C. Nitrous oxide reductase from denitrifying Pseudomonas stutzeri—genes for copper-processing and properties of the deduced products, including a new member of the family of ATP/GTP-binding proteins. Eur. J. Biochem. 192, 591–599 (1990).
CAS PubMed Article Google Scholar
Zhang, L., Wüst, A., Prasser, B., Müller, C. & Einsle, O. Functional assembly of nitrous oxide reductase provides insights into copper site maturation. Proc. Natl Acad. Sci. USA 116, 12822–12827 (2019).
CAS PubMed PubMed Central Article Google Scholar
Zumft, W. G. Biogenesis of the bacterial respiratory CuA, Cu–S enzyme nitrous oxide reductase. J. Mol. Microbiol. Biotechnol. 10, 154–166 (2005).
CAS PubMed Google Scholar
Wunsch, P. & Zumft, W. G. Functional domains of NosR, a novel transmembrane iron-sulfur flavoprotein necessary for nitrous oxide respiration. J. Bacteriol. 187, 1992–2001 (2005).
CAS PubMed PubMed Central Article Google Scholar
Bennett, S. P. et al. NosL is a dedicated copper chaperone for assembly of the CuZ center of nitrous oxide reductase. Chem. Sci. 10, 4985–4993 (2019).
CAS PubMed PubMed Central Article Google Scholar
McGuirl, M. A., Bollinger, J. A., Cosper, N., Scott, R. A. & Dooley, D. M. Expression, purification, and characterization of NosL, a novel Cu(II) protein of the nitrous oxide reductase (nos) gene cluster. J. Biol. Inorg. Chem. 6, 189–195 (2001).
CAS PubMed Article Google Scholar
Okuda, S. & Tokuda, H. Lipoprotein sorting in bacteria. Annu. Rev. Microbiol. 65, 239–259 (2011).
CAS PubMed Article Google Scholar
Ciccarelli, F. D., Copley, R. R., Doerks, T., Russell, R. B. & Bork, P. CASH—a β-helix domain widespread among carbohydrate-binding proteins. Trends Biochem. Sci. 27, 59–62 (2002).
CAS PubMed Article Google Scholar
Lee, J. Y. et al. Crystal structure of the human sterol transporter ABCG5/ABCG8. Nature 533, 561–564 (2016).
ADS CAS PubMed PubMed Central Article Google Scholar
Thomas, C. & Tampé, R. Multifaceted structures and mechanisms of ABC transport systems in health and disease. Curr. Opin. Struct. Biol. 51, 116–128 (2018).
CAS PubMed Article Google Scholar
Rees, D. C., Johnson, E. & Lewinson, O. ABC transporters: the power to change. Nat. Rev. Mol. Cell Biol. 10, 218–227 (2009).
CAS PubMed PubMed Central Article Google Scholar
Bi, Y. C., Mann, E., Whitfield, C. & Zimmer, J. Architecture of a channel-forming O-antigen polysaccharide ABC transporter. Nature 553, 361–365 (2018).
ADS CAS PubMed PubMed Central Article Google Scholar
Qian, H. W. et al. Structure of the human lipid exporter ABCA1. Cell 169, 1228–1234 (2017).
CAS PubMed Article Google Scholar
Diederichs, K. et al. Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis. EMBO J. 19, 5951–5961 (2000).
CAS PubMed PubMed Central Article Google Scholar
Nguyen, P. T., Lai, J. Y., Lee, A. T., Kaiser, J. T. & Rees, D. C. Noncanonical role for the binding protein in substrate uptake by the MetNI methionine ATP binding cassette (ABC) transporter. Proc. Natl Acad. Sci. USA 115, E10596–E10604 (2018).
CAS PubMed PubMed Central Google Scholar
Oldham, M. L., Khare, D., Quiocho, F. A., Davidson, A. L. & Chen, J. Crystal structure of a catalytic intermediate of the maltose transporter. Nature 450, 515–521 (2007).
ADS CAS PubMed Article Google Scholar
Manolaridis, I. et al. Cryo-EM structures of a human ABCG2 mutant trapped in ATP-bound and substrate-bound states. Nature 563, 426–430 (2018).
ADS CAS PubMed PubMed Central Article Google Scholar
Banci, L., Bertini, I., Del Conte, R., Markey, J. & Ruiz-Duenas, F. J. Copper trafficking: the solution structure of Bacillus subtilis CopZ. Biochemistry 40, 15660–15668 (2001).
CAS PubMed Article Google Scholar
Culotta, V. C. et al. The copper chaperone for superoxide dismutase. J. Biol. Chem. 272, 23469–23472 (1997).
CAS PubMed Article Google Scholar
Prasser, B., Schöner, L., Zhang, L. & Einsle, O. The copper chaperone NosL forms a heterometal site for Cu delivery to nitrous oxide reductase. Angew. Chem. Int. Edn Engl. 60, 18810–18814 (2021).
CAS Article Google Scholar
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).
CAS PubMed Article Google Scholar
Ritchie, T. K. et al. Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol. 464, 211–231 (2009).
CAS PubMed PubMed Central Article Google Scholar
Chifflet, S., Torriglia, A., Chiesa, R. & Tolosa, S. A method for the determination of inorganic phosphate in the presence of labile organic phosphate and high concentrations of protein—application to lens ATPases. Anal. Biochem. 168, 1–4 (1988).
CAS PubMed Article Google Scholar
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
PubMed Article Google Scholar
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
CAS PubMed PubMed Central Article Google Scholar
Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).
CAS PubMed PubMed Central Article Google Scholar
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).
PubMed PubMed Central Article Google Scholar
Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).
ADS CAS PubMed PubMed Central Article Google Scholar
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
PubMed PubMed Central Article Google Scholar
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
CAS PubMed Article Google Scholar
Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ 6, 5–17 (2019).
CAS PubMed PubMed Central Article Google Scholar
Scheres, S. H. W. Processing of structurally heterogeneous cryo-EM data in RELION. Methods Enzymol. 579, 125–157 (2016).
Bai, X. C., Rajendra, E., Yang, G. H., Shi, Y. G. & Scheres, S. H. W. Sampling the conformational space of the catalytic subunit of human γ-secretase. eLife 4, e11182 (2015).
PubMed PubMed Central Article Google Scholar
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
CAS PubMed PubMed Central Article Google Scholar
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
CAS PubMed PubMed Central Article Google Scholar
Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
CAS PubMed Article Google Scholar
Goddard, T. D., Huang, C. C. & Ferrin, T. E. Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287 (2007).
CAS PubMed Article Google Scholar
Zhang, L., Trncik, C., Andrade, S. L. & Einsle, O. The flavinyl transferase ApbE of Pseudomonas stutzeri matures the NosR protein required for nitrous oxide reduction. Biochim. Biophys. Acta 1858, 95–102 (2017).
CAS Article Google Scholar
Dell’Acqua, S. et al. Electron transfer complex between nitrous oxide reductase and cytochrome c552 from Pseudomonas nautica: kinetic, nuclear magnetic resonance, and docking studies. Biochemistry 47, 10852–10862 (2008).
CAS PubMed Article Google Scholar
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
ADS CAS PubMed PubMed Central Article Google Scholar
Download references
This work was supported by the European Research Council (grant no. 310656) (O.E.) and Deutsche Forschungsgemeinschaft (CRC 1381, project ID 403222702, and RTG 2202, project ID 46710898) (O.E.) and the BIOSS Centre for Biological Signalling Studies at Albert-Ludwigs-Universität Freiburg (O.E.), and a McKnight Scholar Award (J.D.), a Klingenstein-Simon Scholar Award (J.D.), a Sloan Research Fellowship in neuroscience (J.D.), a Pew Scholar in the Biomedical Sciences award (J.D.) and a NIH grant (R01NS111031) (J.D.). We thank K. Locher for discussions and insights; G. Zhao and X. Meng for support with data collection at the David Van Andel Advanced Cryo-Electron Microscopy Suite; and H. Scott of the electron microscopy facility at the Pacific Northwest Center for Cryo-EM (PNCC) for assistance with data collection. We acknowledge the high-performance computing team of Van Andel Institute and the bwHPC Cluster of the federal state of Baden-Württemberg and the Deutsche Forschungsgemeinschaft (grant INST 35/134-1 FUGG) for computational support. We thank E. Haley, W. Choi, Z. Ruan and Y. Huang in the Du and Lü labs for help with cryo-EM experiments.
These authors contributed equally: Christoph Müller, Lin Zhang
Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany
Christoph Müller, Lin Zhang, Sara Zipfel, Annika Topitsch, Marleen Lutz, Johannes Eckert, Benedikt Prasser & Oliver Einsle
BioEM Lab, Biozentrum, Universität Basel, Basel, Switzerland
Mohamed Chami
Department of Structural Biology, Van Andel Institute, Grand Rapids, MI, USA
Wei Lü & Juan Du
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
C.M., L.Z. and O.E. designed the experiments. C.M., L.Z., S.Z., M.L., A.T., B.P., M.C. and J.E. performed the experiments, C.M., L.Z., S.Z., W.L., O.E. and J.D. processed data. C.M., L.Z. and S.Z. built and refined the structural model. C.M., L.Z., J.D. and O.E. wrote the manuscript.
Correspondence to Juan Du or Oliver Einsle.
The authors declare no competing interests.
Nature thanks Sofia Pauleta, Markus Seeger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, The copper-dependent enzyme N2OR assembles a composite active site at the homodimer interface, consisting of the mixed-valent [Cu1.5+:Cu1.5+] electron transfer centre CuA in one monomer, and the unique, tetranuclear CuZ cluster with composition [4Cu:2S] in the other monomer. Substrate access and binding occurs between both clusters6. b, The Nos system uses the ABC transporter NosFY in conjunction with the periplasmic protein NosD, as well as the dedicated copper chaperone NosL, which was described until now as an outer membrane lipoprotein, whereas the ascribed role of NosD was in sulfur delivery to N2OR. In addition, the nos operon contains the putative quinol oxidase NosR that requires the NosX protein (in P. stutzeri replaced by AbpE57) and may transfer electrons to N2OR. Alternatively, a soluble cytochrome c552 was suggested as physiological electron donor to the enzyme58. c, The nos machinery of P. stutzeri is encoded in a gene cluster of the structure nosRZDFYLtatE.
SEC profiles in DDM micelles and nanodiscs of NosDFY and NosDF(E154Q)Y and the complexes NosDFY with NosL and NosDF(E154Q)Y with NosZ, together with the corresponding analyses by SDS-PAGE. All purifications were performed at least three times with highly reproducible outcomes. The figure shows representative SEC profiles and SDS PAGEs.
a, NosD, coloured from blue at the N terminus to red at the C terminus (marked). Helices hI, hII and hIII interact with NosY. b, The ATPase domain NosF with the C-terminal R domain. c, The transmembrane domain NosY, with the second protomer coloured in white. d, In the NosF dimer, the R domains are crossed, providing additional stability. e, In the nucleotide-free state, the R domain was found in an ensemble of conformations that could be individually refined and revealed a high degree of structural flexibility. f, Superposition of NosD (green) with its closest structural relative, the CASH family protein DFA-IIIase (PDB 5ZKS, r.m.s.d. = 2.68 Å). g, NosF (light orange) and the related ABC domain MalK of the E. coli maltose importer (PDB 3RLF, r.m.s.d. = 2.05 Å). Note that although MalK contains an additional C-terminal domain, it shows no relevant similarity to the R domain. h, NosY (red) and the TMD of the Wzm/Wzt O-antigen transporter (PDB 6M96, r.m.s.d. = 6.0 Å).
a, The maps section shows a stereo representation of the electron density map around the HMM motif of NosD as indicated in the cartoon of the complex. In the ATP-bound state of the NosF(E154Q) variant complex, the HMM motif (residues H297, M209, M231) are metal-free. b, Electron density map for the NosDFYL complex in GDN micelles, showing the same region as in (a). Zn2+ binds to NosL and Cu+ is coordinated by the HMM motif of NosD. c, As the NosZDFY complex was formed with the NosF(E154Q) variant that produces a NosDFY complex that cannot receive Cu from NosL, the metal sites of the apo enzyme are vacant. Electron density map at the CuA site of N2OR. The structure of the holo form of the enzyme (right) underlines the absence of the two Cu ions. d, Electron density map at the CuZ site in the hub of the β-propeller domain of N2OR. The Cu-replete protein in the same orientation (right) denotes where the tetranuclear cluster should be located. All maps are normalized and contoured at the 5σ level.
a, Stereo representation of the C-terminal Mg2+-binding loop in the nucleotide-free state of the NosDFY complex. The cation is octahedrally coordinated in the region from residues D359 to D367, orienting arginine R360 to tightly interact with the membrane-integral NosY subunit. b, Cartoon of NosD bound to the open NosY dimer in the nucleotide-free state of NosDFY. The HMM motif is indicated by a violet disc. c, Cartoon of NosD bound to the closed NosY dimer in the ATP-bound NosF(E154Q) variant.
a–c, Structural asymmetry in the NosY dimer induced by binding of NosD. Top views, coloured by relative root-mean-squared deviations of atom positions. a, Nucleotide-free NosFY subcomplex with symmetric protomers. b, Nucleotide-free NosDFY complex. Major distortions are observed on the periplasmic side of the right protomer, in TM helix 5 and the absence of subhelix 5c (green circle in (a)) owing to structural disorder. c, ATP-bound structure of NosDF(E154Q)Y. As the NosY protomers close, TM helix 5 moves outward, but the structural asymmetry remains, including the disorder of helix 5c in the right protomer. d, Intramembrane modules for P. stutzeri NosY, H. sapiens ABCG5/G8, H. sapiens ABCA1 and A. aeolicus Wzm in cartoon representation. For each system, one subunit of the TMDs is coloured from blue (N-terminus) to red (C terminus). e, Following the topology of NosY, the individual transmembrane helices and the small reentrant helices 5a, 5b and 5c are shown in their relative orientation within the membrane with representative EM density maps, contoured at the 5σ level.
a, Sequence alignment for the HMM motif in NosD proteins. The histidine and two methionine residues are among the most highly conserved in NosD. b, ATP-hydrolytic activity of Nos(D)FY(L) complexes. Substrate dependence of ATPase activity for NosFY, NosDFY, NosDFYL and the NosD(E154Q) variant of the NosDFY complex in DDM micelles. c, Comparison of ATPase activity at [ATP] = 1 mM for protein preparations reconstituted in DDM micelles or MSP nanodiscs. Note that reconstitution of the NosD(E154Q) variant in nanodiscs did not yield a good SEC profile and was therefore not considered successful (Extended Data Fig. 2). n = 3 technically independent samples. Bars are represented as mean values ± SD. d, Data table for (b). e, The human transporter ABCA1, or cholesterol efflux regulator protein (CERP). Cryo-EM structure of human ABCA1, depicted in an open state32. The entire transporter is a single polypeptide, and the extensive extracellular domain is composed of insertions into both transmembrane subunits. In the structural model, the prominent R domains are not swapped between the two halves of the transporter. e, Human ABCA1 as predicted by AlphaFold259. The transmembrane domains are highly similar to the closed state of NosDFY in the NBDs and TMDs. Note that in this model the R domains are swapped, as is the case in NosDFY. f, Conformational differences in the extracellular domain of ABCA1 between cryo-EM structure and AlphaFold2 prediction, in front and top view, respectively. As in NosD, the domain undergoes a rotational motion upon closure of the transporter that is most pronounced in the apical domain.
a, Residue C48(NosL) bridges the Zn2+– and Cu+-binding sites of the chaperone. Its removal renders the chaperone inactive, as evidenced by the failure to mature either Cu site of N2OR at low external Cu. b, Residue P393(NosD) is in the NosD–NosY interface, at the N-terminus of helix hIII. Its replacement for a bulky Trp largely, but not fully impairs the assembly of CuZ. Note that the maturation assay was carried out at high Cu concentrations to have direct CuA assembly by Cu2+ as a positive control. c, Residue V407(NosD) marks the C-terminal end of helix hIII. Disturbing this interaction renders NosDFY fully non-functional. d, The lid loop of NosD with residue M279(NosD) is not highly conserved among NosD proteins. Accordingly, its deletion impairs, but not prevents CuZ maturation at low copper concentration (middle). At high Cu concentrations, N2OR maturation is intact, pointing towards a role of the lid loop in stabilizing Cu bound to NosD.
This file contains the Supplementary Discussion including the following sections: nitrous oxide reductases belong to two distinct clades; AMPPNP-bound NosDFY does not switch to the closed state; localization of NosL to the inner membrane; a back-and-forth of entropy and conformational changes in NosDFY and the analogy to ABCA1. It also contains Supplementary Figs. 1–9.
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
Reprints and Permissions
Müller, C., Zhang, L., Zipfel, S. et al. Molecular interplay of an assembly machinery for nitrous oxide reductase. Nature (2022). https://doi.org/10.1038/s41586-022-05015-2
Download citation
Received: 06 April 2021
Accepted: 23 June 2022
Published: 27 July 2022
DOI: https://doi.org/10.1038/s41586-022-05015-2
Anyone you share the following link with will be able to read this content:
Sorry, a shareable link is not currently available for this article.
Provided by the Springer Nature SharedIt content-sharing initiative
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
Advertisement
Advanced search
© 2022 Springer Nature Limited
Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.