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Molecular interplay of an assembly machinery for nitrous oxide reductase

Abstract

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.

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Fig. 1: Three-dimensional structure of P. stutzeri NosDFY in the nucleotide-free state.
Fig. 2: Conformational dynamics of NosDFY and copper binding to NosD.
Fig. 3: Nucleotide-free NosDFY receives Cu+ from NosL.
Fig. 4: Interaction of the apo enzyme with the ATP-bound state of NosDFY.
Fig. 5: Model for copper trafficking from NosL via NosDFY to N2OR.

Data availability

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.

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Acknowledgements

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.

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Contributions

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.

Corresponding authors

Correspondence to Juan Du or Oliver Einsle.

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Extended data figures and tables

Extended Data Fig. 1 The nitrous oxide reductase (nos) system.

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.

Extended Data Fig. 2 SEC profiles in DDM micelles and nanodiscs.

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.

Extended Data Fig. 3 Structural details of NosDFY.

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).

Extended Data Fig. 4 Exemplary electron density maps of NosD.

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.

Extended Data Fig. 5 The Mg2+-binding site and conformations of NosD.

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.

Extended Data Fig. 6 The transmembrane module belongs to type-IV ABC transporters (type-II exporters).

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.

Extended Data Fig. 7 Properties of NosDFY complexes and related transporters.

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.

Extended Data Fig. 8 Variant studies on the interactions of NosD.

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.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Extended Data Table 2 Cryo-EM data collection, refinement and validation statistics (continued)

Supplementary information

Supplementary Information

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.

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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

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