Structural insights into the catalytic mechanism of ammonia monooxygenase

Domain: physics
Published: 2025-12-12
Editorial outcome: major_revision

Abstract
Ammonia monooxygenase (AMO) oxidizes ammonia to hydroxylamine. Limited knowledge of the structural information of AMO hinders our understanding of the molecular mechanism underlying ammonia oxidation, impacting the mitigation of greenhouse gas emissions and enhancing agricultural productivity using ammonium as a nitrogen source. Herein, we report the cryo-electron microscopy structure of the AMO complex from an isolated strain of ammonia-oxidizing bacteria (AOB). AMO is a cylinder-shaped homotrimeric assembly composed of five subunits. A single-transmembrane protein and a soluble protein are potentially crucial in signal transduction during ammonia oxidation and mediating interactions with the outer membrane protein assembly machinery. Three modeled coppers, along with an adjacent water-mediated hydrogen-bond network, may facilitate an efficient proton transfer pathway from the periplasmic Cu to the active site Cu within the inner membrane, where Cu and Cu will act in concert to catalyze substrate reaction. The distinctive surface charge characteristics of AMO provide valuable insights into the structural features that govern ammonium assimilation and material transport during ammonia oxidation. These findings shed light on the molecular complexities of AMO and provide a structural foundation for elucidating the catalytic mechanism of ammonia oxidation.

Introduction
Ammonia oxidation (AO), the pivotal and rate-determining step in nitrification, holds a crucial position in the global nitrogen cycle. This process is primarily orchestrated by ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and complete ammonia oxidizers commonly referred to as comammox1–4. These three types of ammonia oxidizers exhibit distinct variations in their environmental distributions due to differences in substrate affinity5,6. In addition, AOB produce higher levels of nitrous oxide (NO) than AOA both in natural environments and in pure cultures7–10, thereby exerting a significant impact on global warming.

The initial step in AO entails the oxidation of ammonia into hydroxylamine (NH OH), a process catalyzed by copper-dependent ammonia monooxygenase (AMO)11 through the reaction NH +O +2e-+2H+→NH OH+H O12. Biochemical data have demonstrated that the AMO core is composed of AmoA, AmoB and AmoC13–15. Subsequently, two genes (orf1 and orf5) located downstream of the amoCAB gene cluster were regarded as accessory components of the AMO in AOB16. AMO in AOA has recently been identified as a complex consisting of six subunits (amoABCXYZ), exhibiting only a 40% sequence similarity to their bacterial counterparts, underscoring the unexpected complexity of this enzymatic complex17. More recently, Tucci et al. reported the 2.8 Å structure of AMO from Nitrosomonas europaea and discovered a single transmembrane protein subunit18. However, the enzymatic mechanism of AMO and the identity and function of its cofactors remain largely enigmatic due to the limitation of resolution and the deficiency of the active Cu site.

The particulate methane monooxygenase (pMMO), a key enzyme in carbon transformation and a member of the membrane monooxygenase family alongside AMO, is responsible for methane oxidation19–22. Copper ligands are indispensable to the catalytic activity of AMO and pMMO11,23. Over two decades of structural investigations have unveiled the α3β3γ3 trimeric arrangement of pMMO, consisting of three components—PmoA, PmoB and PmoC. While several copper ligand environments have been identified in pMMO, the identity of the authentic active site has long been a subject of debate22. The bis-His site (Cu ) has historically been considered unlikely to be the active center due to the lack of conservation in coordinating residues24. Previous extended X-ray absorption fine structure (EXAFS) data indicated a dinuclear feature of Cu-Cu scattering interaction in Cu site25,26. Subsequently, the high-resolution structures showed a mononuclear Cu site27–30. Cu presents as Cu(II)31 and has no obvious hydrophobic pocket for substrate binding. Further, the mutation of one of the Cu ligands in a related hydrocarbon monooxygenase did not completely abolish activity32. Consequently, scientists currently tend to believe that the Cu site is not an active site. The earlier crystal structures revealed that zinc ions occupy the Cu site due to the artificial addition of crystallization reagents ZnSO 24,27. However, its strict conservation and completely abrogated activity upon mutation of coordinated residues suggest that the Cu is the likely active site32. Recently, the presence of the Cu site, the binding of a substrate analogue near the Cu and the electron nuclear double resonance data supported the notion that the Cu is the active site33,34. However, quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations suggest that both Cu and Cu tend to remain in the Cu(I) active state35, highlighting the potential relevance of a dicopper state. Given that the short distance between them (~5.7 Å resolved in previous pMMO structures containing single Cu or Cu ), the possibility of co-occupied Cu and Cu sites remains open22. In short, fundamental inquiries persist concerning the precise locales of substrate oxidation under the cooperative influence of these copper ions.

Here, we have determined the 2.36 Å structure of AMO from an isolated AOB by leveraging the cryo-EM single particle analysis. This structure revealed a homotrimeric assembly of the heteropentamers AMO complex, each composed of five distinct components. Notably, the recently discovered single transmembrane protein AmoD18 and an identified component AmoE were resolved to side-chain resolution, offering insights into their potential roles in signal transduction and interactions with the outer membrane protein assembly machinery during AO. Moreover, extensive lipid densities were observed within the AMO structure, with their distribution, shape, and arrangement closely mirroring the native membrane environment, suggesting that AMO particles are likely preserved in their native functional state. In addition, the high-resolution structure revealed the coordination environments of the three copper ions within the AMO, along with an adjacent water-mediated hydrogen-bond network, which may facilitate an efficient proton transfer pathway from Cu to the co-occupied copper site (Cu and Cu ), where the potential intermediate density was identified. Furthermore, the surface electrostatic potentials of AMO imply potentially divergent mechanisms governing the binding and delivery of various substrates and products for AMO and pMMO.

Results
Isolation of AOB and purification of AMO
Chemolithotrophic AOB serve as a pivotal architect in the global nitrogen cycle, orchestrating the transformation of natural ammonia into nitrite1,36. The initial step of nitrification, wherein ammonia is oxidized to hydroxylamine, is catalyzed by the integral membrane-embedded AMO complex11,37,38. To delve deeper into the structural insights of AMO within AOB, we successfully enriched and isolated a pure culture of AOB (Nitrosomonas halophila ZJB1) from mangrove sediment using traditional enrichment techniques (Supplementary Fig. 1). This identified subspecies, classified within the N. europaea/Nitrosococcus mobilis lineage based on 16S rRNA phylogenetic analysis, exhibits high enzymatic activity for AO (Supplementary Fig. 1a). We engineered a genetic manipulation system that introduced a high-specificity fusion peptide (3×Flag) tag to the C-terminus of AmoA to affinity purify the high-quality samples of the AMO complex (Supplementary Fig. 1b). Following this, a gentle yet efficient purification protocol yielded monodispersed and uniform AMO complexes solubilized in the mild detergent digitonin (DIG) (Supplementary Fig. 2). Enzymatic assays were performed using electron donors commonly applied in studies of pMMO to assess AO activity of the purified AMO sample. However, no detectable AMO activity was observed under these conditions (Supplementary Fig. 3a, b). Through screening potential redox-active compounds, we identified ubiquinol (CoQ H ) as a candidate electron donor that moderately enhanced AMO activity (Supplementary Fig. 3c, d). In addition, a significant decline in activity was observed concomitant with the progressive loss of native cellular integrity (Supplementary Fig. 3e), which is not caused by disruption of the AMO structure, as compared to the recently reported AMO structure in the native membrane18 (vide infra). This result suggests that efficient AO relies on factors beyond CoQ H alone. Importantly, the AO activity of the purified AMO sample was compared with that of the membrane-bound form, indicating that the purified AMO retained substantial enzymatic activity (Supplementary Fig. 3f). Then, these freshly isolated AMO samples were subsequently subjected to single-particle cryo-EM analysis, resulting in a high-resolution 3D reconstruction map with a nominal resolution of 2.36 Å (Supplementary Fig. 4 and Supplementary Table 1). This resolution is adequate for unambiguously building an atomic model of AMO at the side-chain level with high confidence (Fig. 1 and Supplementary Fig. 5).

Overall structure of AMO
The overall architecture of AMO exhibits a cylindrical shape with a volume of ~105 Å x 105 Å x 115 Å. AMO oligomerizes as a homotrimer of heteropentamers. Each protomer consists of AmoA, AmoB, AmoC, and a recently discovered single transmembrane protein and an identified subunit, hereafter referred to as AmoD and AmoE (distinct from the previously predicted AmoD and AmoE39 (vide infra) (Fig. 1). Two β-barrel domains from the N-terminus and C-terminus of AmoB interact in an almost perpendicular orientation, arranged in a counterclockwise manner as a trimer, forming the periplasmic regions of AMO (Fig. 1). This structure is supported by 45 transmembrane (TM) helices, with contribution of six from AmoA, two from AmoB, six from AmoC, and one from AmoD in each protomer respectively (Fig. 1b). Similar to the pMMO, the intracellularly soluble region of AMO is primarily composed of AmoA, AmoB and AmoC, forming a triangular scaffold bridged by the extended C-terminus of AmoA. A central pore spans the entire AMO complex and is lined with a bilayer of lipids (Fig. 1a). In accordance with the findings from the pMMO nanodisc system33, extensive potential lipid densities were discovered within the central pore and on the surface of the TM region (Fig. 1a and Supplementary Fig. 6a), suggesting that the native cellular environment is preserved during protein purification. To identify the specific lipid identities present in the sample, we conducted a comprehensive lipidomics analysis. In contrast to the predominant lipid composition in intact bacterial cells, the major lipid species identified in the AMO sample were free fatty acid (FFA), monoglyceride (MG), diglyceride (DG), and phosphatidylserine (PS) (Fig. 1d and Supplementary Fig. 6b, c), suggesting a selective enrichment of lipids that interact with AMO. By integrating the spectrometric evidence with the cryo-EM density maps, we ultimately incorporated 84 lipid molecules into the AMO structure (Fig. 1e). These lipids were predominantly distributed across three regions.

One cluster of lipids resembling a plug is situated at the upper part of the central pore, with its head groups facing the periplasm and acyl chains stretching into the inner pore (Supplementary Fig. 6a, left panel). Near the cytoplasmic region of AMO, a propeller-shaped lipid buckles the outlet of the pore. Compared to the lipids in the upper part of the central pore, these lipids are arranged in an opposing fashion, with their polar head groups directed toward the cytoplasm. Lipids from two adjacent AMO monomers form three blade-like structures (Supplementary Fig. 6a, right panel). In the transmembrane region, a bilayer of lipids is observed, with their hydrophobic acyl chains aligned along the equatorial plane of the plasma membrane (Supplementary Fig. 6a, middle panel). Collectively, the distribution, shape and arrangement of aforementioned of these lipids closely mirror the native membrane environment.

Copper centers and proton transfer pathway in AMO
Similar to pMMO, AMO has been established as a copper-dependent oxidase11. Extensive structural data have previously shown diverse copper centers in pMMO, as summarized in Supplementary Fig. 7. The absence of the proposed active Cu site in the recently reported AMO structure18 leaves the exact copper centers and active site in the AMO unresolved to date. The high-resolution structure of the AMO resolved in this study enabled the identification of nine metal-like density regions within the trimeric AMO, positioned at sites analogous to the Cu , Cu and Cu binding sites in pMMO. Each AMO protomer contains three copper ligands that form two distinct copper centers (Fig. 2a). The first copper center resides within the N-terminal β-barrel of AmoB and is coordinated by H38, H142 and H144 (Fig. 2b), a configuration conserved across all observed pMMO (Supplementary Fig. 7) and the recently reported AMO18. Consequently, it is designated as the Cu site. Despite the conservation of these coordinating residues, the number of copper ions at this site has been a subject of debate. For instance, dinuclear copper centers are observed in crystal and cryo-EM structures of pMMO from several species24,27,40,41, whereas many other structures exhibit mononuclear copper28,33,34 (Supplementary Fig. 7). In the high-resolution AMO resolved in this study, the Cu center unambiguously displays a mononuclear copper (Fig. 2b). Consistent with crystallographic and spectroscopic studies of Cu 28,29,31,42, an axial water molecule positioned below the Cu site forms a weak interaction with Cu at a distance of 3.9 Å. In contrast to the individual Cu and Cu sites previously observed in pMMO structure and the recently resolved AMO, the second copper center in our AMO structure is unexpectedly characterized by two simultaneously occupied copper ligands—Cu and Cu (Fig. 2c–e). This prominent feature is reminiscent of the recent QM/MM MD result that both Cu and Cu tend to remain in Cu(I) active state35, suggesting the potential synergistic effect of Cu and Cu on AO. Examination of the neighboring environment around the second copper center reveals that five TM helices—TM1 from AmoA and TM3, TM4, TM5 and TM6 from AmoC—converge to form a hydrophobic channel. This channel cradles the dinuclear copper center at its apex (Fig. 2c, h).

In contrast to the single copper center in pMMO and AMO, TM5 of AmoC from the dinuclear copper center resolved in this study is bifurcated into two smaller helices. The upper helix obviously shifts towards the central channel, thereby expanding the volume of this hydrophobic channel to accommodate the dinuclear copper ligands (Fig. 2h). The observed change in the diameter of this hydrophobic channel with an increase in the number of copper ligands may reflect regulation of AMO activity, and the TM5 of AmoC may play a crucial role in this process. Sequence alignment demonstrates that TM5 is conserved among AOB and methane-oxidizing bacteria (MOB). Notably, the histidine and asparagine residues that coordinate Cu , as well as the residues at the kink site, are highly conserved (Fig. 2i), indicating a shared role in regulating the Cu site and hydrophobic channel in both AMO and pMMO. Cu establishes a stable coordinated environment with two positively-charged residues (H140 and H153) and two negatively-charged residues (D 136 and D146) (Fig. 2d). It is worth mentioning that Asp146 exhibits less conservation and may be replaced by Arginine/Glutamate33,34,41, or even be absent in pMMO27,28,33. Analogous to the Cu site, a spherical density approximately 3.3 Å from the Cu site was observed and modeled as a water molecule (Fig. 2c). Cu , recently identified as a potential active site in pMMO34, engages in an interaction network through H211, H225, N207 and E218 (Fig. 2e). E218 was absent in the pMMO structure (Supplementary Fig. 7). To probe the functional relevance of these metal-coordinating residues, we constructed single-copy mutations in H140A, H153A, D146A, N207A, E218A, and H211A. Attempts to generate D136A and H225A mutants were unsuccessful, likely owing to the severe functional defects caused by these substitutions. Enzyme activity assays revealed that most mutations resulted in an approximately 30% reduction in activity (Fig. 2g), comparable to the effect caused by the deletion of single AMO subunits43. Given the presence of three copies of the amo operon in the genome, these findings underscore the essential role of these residues in AO.

Close inspection of the coordination environments of the three coppers revealed the consistent presence of a relatively weak hydrogen bond associated with these sites (Fig. 2b, d, e, red stars), suggesting their potential role in facilitating proton transfer during substrate oxidation, beyond merely stabilizing the copper sites. Between the Cu and Cu ligands, we observe two distinct densities (Fig. 2c). While these are tentatively modeled as water molecules based on precedents in related metalloenzyme structures18,27,33, we cannot exclude the possibility that they represent oxygen species derived from dioactivation. The dinuclear nature of this center invites comparison to type III copper sites involved in O binding and activation44; however, the longer metal–metal distance and atypical coordination geometry suggest a distinct mechanism in AMO (Supplementary Fig. 8). Recent studies have proved that Cu serve as the active site for substrate oxidation in pMMO, with the substrate binding site located in proximity to Cu 33,34. Interestingly, an elongated, rod-shaped density was observed in the hydrophobic channel adjacent to the Cu –Cu center (Fig. 2c). While its identity remains uncertain, its location suggests a potential role in substrate access or product release. Further studies at higher resolution or under different catalytic conditions are necessary to elucidate the nature of this feature.

Integrating the aforementioned structural features, it appears more plausible that the co-occupied copper center represents the genuine active site of AMO, functioning in a manner analogous to a type III copper center, despite the presence of certain distinct catalytic characteristics (Supplementary Fig. 8)45. In this context, the metalloenzyme utilizes a pair of copper ligands as its catalytic active center to facilitate the catalytic process. Based on the Grotthus mechanism46, we discovered an extensive water-mediated hydrogen-bonding network from Cu to the Cu and Cu site. The hydrogen bond distances, ranging from 3.9 Å (near the Cu ) to 2.6 Å (near the Cu ), may reveal an efficient proton transfer to the co-occupied Cu and Cu active center to facilitate the AO reaction, as indicated by the high-resolution structure of photosystem II47–49 (Fig. 2f). However, further experimental evidence is required to substantiate this proposal. In addition, the bis-His site is not present in the AMO structure due to the substitution of the first Histidine by Glutamine in the AmoB sequence, as observed in the pMMO structure of Methylocystis sp. str. Rockwell, suggesting less conservation of the bis-His site.

AmoD and the identified AmoE
Over the years, extensive efforts have been dedicated to elucidating the structure and composition of AMO by integrating methodologies from genomics, biochemistry, bioinformatics and biophysics. In addition to three proposed components based on the homology to pMMO—AmoA, AmoB and AmoC50, scientists have recently uncovered evidence suggesting the presence of additional components within AMO17. However, the precise architecture remains enigmatic due to the limitation of the direct structure. Beyond the aforementioned three core subunits, the structure of AMO resolved in this study reveals a recently reported single transmembrane protein (identified as NZ4318 in this study)18 and an identified component (NZ3624) at the periphery of AMO based on the high-resolution structure at the side-chain level (Supplementary Fig. 5), assigned as AmoD and AmoE, respectively.

These proteins diverge from the earlier predicted AmoX/Y/Z in archaeal AMO17,51. In contrast, AmoD is relatively conserved with the addition of a reported transmembrane helix18 (Supplementary Fig. 9), indicating a compositional difference between bacterial and archaeal AMO. AmoD is positioned adjacent to AmoC with its N-terminal facing the cytoplasmic side and its C-terminal oriented towards the periplasmic side (Figs. 1 and 3a). A similarly enigmatic helical structure has been observed in pMMO, but efforts to elucidate its identity were impeded by inadequate resolution27,28,40. In line with the crystal structure of Methylocystis sp. str. Rockwell pMMO, a robust density of phospholipid, was modeled near the AmoD. The conformation of this phospholipid differs from that of pMMO, as it is fully embedded in the hydrophobic interior surface of AmoD and AmoC. The lipid species was identified as phosphatidylmethanol (PMeOH) by integrating the lipidomic data with the corresponding density. The phosphate group of PMeOH interacts with R178, a conserved residue from the AmoC subunit, while its alkyl tails extend into the hydrophobic interface formed by AmoC and AmoD (Fig. 3b). In addition, a second previously undetected phospholipid, identified as diacylglycerol (DG), resides at the interface between the inter-protomer AmoC and the C-terminal of AmoD (Fig. 3c). The first alkyl chain of DG interacts with AmoD through W37 and penetrates the hydrophobic core formed by AmoD and AmoC. Concurrently, the second alkyl chain anchors onto the surface of TM1 in AmoC and extends into the lipid bilayer of the cell membrane. This distinct lipid-binding mode suggests that these interactions may contribute to stabilizing the structural integrity of the AMO complex and facilitating the proper integration and localization of AmoD within the membrane environment.

In contrast to AmoD, the identified protein AmoE is a soluble entity, comprising two small helices (H1 and H2) and two flexible loops (L1 and L2) (Fig. 1c). The L1 and H2 are well defined by high-resolution density (Supplementary Fig. 5). The H2 of AmoE binds to the positively-charged surface of the N-terminal cupredoxin domain of AmoB through electrostatic interactions mediated by residues T165, I164, N163, G139, R140, W161 from AmoB and I37, Y34, E30 from AmoE (Fig. 3d, e). Meanwhile, the L1 of AmoE protrudes into the negatively-charged cleft formed by the periplasmic regions of AmoA and AmoC, as well as the N-terminal cupredoxin domain of AmoB (Fig. 3d). Specifically, R21 of AmoE interacts with F171 and W168 of AmoB. P22 of AmoE simultaneously links to two tryptophan residues from AmoB and AmoA (W168 and W113) via hydrophobic interactions. Q24 of AmoE connects with H116 of AmoA and S100 of AmoB via electrostatic interactions. In addition, L26 and E28 of AmoE form multiple hydrogen bonds with S53, H54 and E62 of AmoC at the interface between AmoE and AmoC (Fig. 3f).

We performed bioinformatics analysis to investigate the potential functional roles of AmoD and AmoE. Interestingly, the phylogenetic tree revealed that amoD is exclusively present in the N. europaea/N. mobilis lineage, with its genomic location associated with certain key enzymes involved in the tricarboxylic acid cycle (TCA) and the synthesis of crucial signal molecules (such as c-di-GMP) (Fig. 3g and Supplementary Fig. 10a, b). We propose that AmoD may play a role in coordinating electron flow from central metabolism to AMO. This model is supported by our identification of ubiquinol (CoQ H )—a metabolic electron carrier regenerated by TCA cycle-derived reducing equivalents—as an effective electron donor for AMO (Supplementary Fig. 3c, d). Therefore, AmoD may facilitate metabolic coupling to maintain AO activity under diverse physiological conditions. In contrast, the genetic neighborhood of amoE encompasses genes encoding components of the ATPase and outer membrane beta-barrel assembly machinery complex (Fig. 3h and Supplementary Fig. 10a, c), suggesting a potential, though still speculative, functional relationship. Given its periplasmic localization, we hypothesize that AmoE may help coordinate interactions between the inner membrane-embedded AMO complex and outer membrane-associated components, possibly contributing to regulatory or substrate delivery processes. This is consistent with the previously predicted interaction between the AMO and the S-layer outside the membrane in archaea17,52. However, further experimental validation is required to elucidate its precise role.

Surface electrostatic potentials of AMO
The assimilation and transformation of ammonium into hydroxylamine play a crucial role, mediated by AMO11. Nonetheless, the precise mechanisms underlying these vital processes within AMO remain elusive. Close inspection of the surface electrostatic potentials of AMO revealed distinctive characteristics that diverge from those of pMMO. Notably, the periplasmic β-barrel domains of AmoB coalesce into three densely negatively-charged regions (DNR), a feature that is absent in pMMO (Fig. 4a, left panel). These DNR may contribute to facilitating the capture of positively-charged ammonium ions from the ambient environment. In addition, two parallel charged zones were distributed along the equatorial plane of the lipid bilayer (Fig. 4a, middle panel). Within the periplasmic layer of lipid, a negatively-charged ribbon, designated as the periplasmic negatively-charged regions (PNR), is situated on the lipid surface adjacent to the extracellular cupredoxin domain of AmoB. Beneath the PNR, a positively-charged ribbon, termed the periplasmic positively-charged regions (PPR), runs parallel spreads and is embedded within the lipid bilayer. Analogously, on the cytoplasmic side, cytoplasmic negatively-charged regions (CNR) and cytoplasmic positively-charged regions (CPR) are arranged in parallel (Fig. 4a, middle panel). These charged regions do not exist at the hydrophobic and hydrophilic interface of the lipid bilayer in pMMO (Fig. 4a, middle panel).

The distinct electrostatic profile at the membrane interface may facilitate the recognition and translocation of ammonium. This architecture is reminiscent of recently reported cation-translocating membrane proteins53, suggesting a conserved mechanism for substrate guidance and uptake. Moreover, unlike the densely charged distribution of the cytoplasmic region of pMMO, AMO exhibits a more evenly distributed charge landscape, attributed to the extended cytoplasmic regions of AmoA, AmoC and AmoD relative to their homologous counterparts PmoA and PmoC (Fig. 4a, right panel and Supplementary Fig. 11). In particular, the AmoA features an elongated C-terminus consisting of a positively-charged β hairpin and a negatively-charged helix. These two structural elements are intricately intertwined in a head-to-tail configuration through robust electrostatic interactions, forming an equilateral triangular scaffold within the cytoplasmic AmoA trimer (Fig. 4b). This architectural arrangement serves as a linchpin in stabilizing the three adjacent AMO protomers and fine-tuning the surface electrostatic distribution of the cytoplasmic regions of AMO. In essence, AMO diverges from pMMO in its surface electrostatic potentials. Given the charged disparities between the respective substrates and products of pMMO and AMO, these distinctive attributes may suggest a potentially divergent mechanism for the binding and delivery of substrates (ammonia, methane, electron donors and oxygen) and products (hydroxylamine and methanol) for AMO and pMMO.

In conclusion, our cryo-EM structure of the AMO complex from N. halophila ZJB1 provides key insights into the molecular architecture and catalytic potential of this environmentally and agriculturally important enzyme. We resolved AMO as a homotrimer of heteropentamers, with each protomer comprising five subunits—AmoA, AmoB, AmoC, AmoD and AmoE—thereby revising the conventional model of its composition. The presence of the single transmembrane protein AmoD and the soluble subunit AmoE suggests roles in structural stabilization, complex assembly, as well as potentially in coordinating cellular metabolism or substrate delivery processes related to AO, supported by their genomic context and localization.

Notably, our structure reveals a dinuclear copper center (Cu -Cu ) with simultaneous occupancy, offering the direct structural evidence for a proposed catalytically relevant state. The adjacent water-mediated hydrogen-bond network suggests a plausible pathway for proton transfer, supporting a mechanism wherein Cu and Cu act in concert to facilitate AO. Furthermore, the electrostatic features at the periplasmic and cytoplasmic interfaces may assist in substrate recruitment and product release, highlighting functional divergence from the related pMMO.

While this study establishes a foundational structural framework, further functional analyses and high-resolution investigations are required to fully elucidate the substrate-binding intermediates, clarify the specific roles of AmoD and AmoE, and delineate the catalytic mechanism of AMO.

Methods
Isolation of pure AOB strain
Surface sediment samples (0–2 cm) were collected from the mud flat in a mangrove wetland in Zhanjiang Bay in November 2020 (Supplementary Fig. 1c). The sediment samples were placed in a cooler and transported to the laboratory for the enrichment and isolation of AOB. Detailed procedures for AOB enrichment and isolation processes are shown in Supplementary Fig. 1a. Briefly, ~2 g of sediment samples were added into 40 mL AOB enrichment medium54, and the resulting mixture was incubated at 30 °C with shaking at 120 rpm in the dark until a high ammonia oxidation rate was detected. Subsequently, a small volume of the enriched culture was transferred to a culture flask containing fresh medium (10% inoculation) for further cultivation until ammonium depletion. The dilution procedure was repeated multiple times until no culturable heterotrophic microorganisms were detected using nutrient agar. The abundances of AOB and total bacteria in these enrichments were quantified based on quantitative PCR of the β-AOB amoA55 and bacterial 16S rRNA genes56, respectively. When AOB accounted for ≥90% of the total bacteria, the enrichment culture was diluted and then inoculated onto AOB solid medium (2% agar)54. A single colony was selected and isolated for further study. Finally, we obtained a pure AOB strain whose 16S rRNA gene sequence exhibited high sequence similarity (>98.8% identity) to that of isolated N. halophila Nm11 and formed a distinct cluster with N. halophila Nm1 in the phylogenetic tree (Supplementary Fig. 1d). Given its sampling location, this pure AOB isolate is named N. halophila ZJB1 (Zhangjiang Bay).

The phylogenetic tree of the AOB 16S rRNA gene
The 16S rRNA gene sequences of representative AOB were retrieved from GenBank (https://www.ncbi.nlm.nih.gov/) for phylogenetic analyses. These sequences were arrayed with MAFFT-L-INS-I57 and pruned using BMGE (BLOSUM30)58. Maximum-likelihood tree was inferred with IQ-TREE under GTR+F+I+G4 model59. Ultrafast bootstrapping (1000 replicates) was used.

Phylogenies of AmoD and AmoE sequences
The AmoE sequence from N. halophila ZJB1 was used as a query to search against the nr database using BLASTP60, and hits with >50% amino acid similarity were downloaded. For AmoD phylogeny, the bacterial sequences containing the lipoprotein family motif of AmoD (PS51257) were downloaded from the Interpro database (https://www.ebi.ac.uk/interpro/search/sequence/). After excluding proteins longer than 100 amino acids, a total of 474 homologous sequences were obtained. These sequences were aligned with MAFFT-L-INS-I57 and pruned using BMGE (BLOSUM30)58. Maximum-likelihood trees were inferred with IQ-TREE (cpREV+G4 for AmoD; WAG+G4 for AmoE; -bb 1000)59.

Chemical analyses
Ammonium and nitrite concentrations were measured using the optimized indophenol blue method61 and the modified Griess-Llosvay procedure62, respectively. Briefly, 100 μL of culture or buffer was aliquoted into sterile 1.5 mL Eppendorf tubes and centrifuged at 15,000×g for 5 min at room temperature to pellet large particulates and cells. The supernatant was transferred to fresh sterile 1.5 mL Eppendorf tubes and diluted to a final volume of 1 mL with NH +-free ultrapure water. For the optimized indophenol blue assay, 50 μL of phenol-nitroprusside solution and 50 μL of alkaline hypochlorite solution were added; alternatively, for the modified Griess-Llosvay assay, 50 μL of sulfanilamide solution and 50 μL of naphthylamine solution were used. Following complete color development, absorbance was measured at 630 nm for ammonium and at 543 nm for nitrite using a microplate reader (EnSpire). Final concentrations were calculated using standard curves for NH +(0–10 μM, n=3, R2>0.998) and NO -(0-20 μM, n=3, R2>0.999), respectively.

DNA extraction, gene cloning, and plasmid construction
DNA was extracted from 2 mL culture using the PowerSoil® DNA Isolation kit (MoBio Laboratories, Carlsbad, CA, USA). The DNA concentrations were measured using the NanoValue™ Plus micro nucleic acid analyzer (GE Healthcare Life, Pittsburgh, PA, USA). The PCR primers used in this study are shown in Supplementary Table 2.
(i) pJTU1278 (Catalog No. TSPLA16579, Ningbo Testa Biotechnology Co., Ltd.), a plasmid used for the construction of a suicide vector harboring the KmR gene. The recombinant suicide plasmid consists of three fragments: the first fragment (2.9 kb) was PCR amplified using the primer pair pJTU-F/pJTU-R, incorporating the ori, lacZ, and AmpR genes from the pJTU1278 plasmid63 along with an XbaI restriction site. The second fragment (558 bp) was PCR amplified using the primer pair oriT-F/oriT-R, containing the conjugative ori and the traJ gene. The third fragment, a kanamycin resistance gene (KmR)64, was synthesized by GENEWIZ Biotechnology Co., LTD (Suzhou, China) and PCR amplified using the primer pair KmR-F/KmR-R, resulting in a 978 bp product that includes the KmR promoter. Subsequently, the three fragments were assembled into the plasmid, pJTU1278-KmR, using the ClonExpress Ultra One Step Cloning Kit V2 (Vazyme, Nanjing, China).
(ii) pJTU1278-KmR, a plasmid used for the assembly of amoCAB genes and fusion tags. A 1.42 kb DNA fragment encompassing the amoC gene and its promoter sequence from N. halophila ZJB1 was amplified by PCR using primers amoC-F/amoC-R, with the reverse primer amoC-R containing a XbaI restriction site and Strep-Tag II fusion tag. The resulting PCR products were purified via gel extraction and ligated into the XbaI site of the vector, followed by transformation into E. coli DH5α cells. Similarly, the amoA (1.08 kb) and amoB (1.28 kb) genes were PCR amplified using respective primer pairs amoA-F/amoA-R and amoB-F/amoB-R. The corresponding PCR products were also gel-purified and ligated into the XbaI site before being transformed into E. coli DH5α cells. In addition, 3×Flag fusion tag was introduced downstream of amoA via reverse primers. Ultimately, we successfully constructed an 8253 bp suicide plasmid, designated as pJTU1278-KmR-AMO.

Electroporation-mediated cell transformation
N. halophila ZJB1 cells (1 L) from an early-stationary-phase liquid culture (OD ≈0.2) were harvested by centrifugation and washed three times with sterile water. The sedimented cells were resuspended in 1.5 mL of sterile water and kept on ice until use. Electroporation was carried out using an ECM399 Electroporation System (BTX Harvard Apparatus, voltage range of 60-2500 V, fixed capacitance of 36 μF, fixed resistance of 150 Ω). Aliquots (100 μL) of electrocompetent cells were transformed with 5 μL plasmid DNA (1 μg DNA/μL) in 1 mm gap parallel electrode cuvettes (Fisherbrand) at a constant voltage of 1.2 kV. A control for the electroporation experiment consisted of cell aliquots without added DNA. Immediately after electroporation, the cells were transferred to 200 mL of fresh medium and incubated for 24 h under nonselective conditions at 30 °C with shaking (100 rpm). Kanamycin was then added to a final concentration of 25 μg/mL. Cell growth was monitored by measuring consumption of NH + and accumulation of NO -. Finally, a small volume of cultures was transferred to a culture flask with fresh medium (10% inoculation) containing kanamycin (25 μg/mL) for further culturing until NH + was depleted. This process was repeated, and after two months, kanamycin-resistant cultures were successfully obtained.

AMO purification
120 L N. halophila ZJB1 cells expressing AmoA-Flag were harvested by vacuum filtration through a 0.22 μM pore polycarbonate membrane filter (Millipore). The collected cells were subsequently resuspended in lysis buffer (50 mM PIPES pH 7.2, 50 mM NaCl, 30 μM CuSO ) and lysed by sonication on ice. The lysate was then supplemented with n-Decyl-β-D-Malyranoside (DDM) to a final concentration of 2%, followed by stirring for 2 h at 4 °C to extract the membrane proteins. Subsequently, cell debris was removed by centrifugation at 20,000×g for 30 mins at 4 °C. The resulting supernatant was collected and incubated with anti-FLAG beads for 2 h at 4 °C. The beads were washed extensively by SEC buffer (50 mM PIPES pH 7.2, 50 mM NaCl, 30 μM CuSO , 0.06% digitonin (DIG)). Proteins were eluted by 200 μg/mL 3×FLAG peptide in the SEC buffer. The eluent was loaded onto a Superose S6 3.2/60 column (GE Healthcare) equilibrated with SEC buffer. Peak fractions were collected for cryo-EM sample preparation.

Activity assays
The ammonia oxidation activity was measured separately using intact AOB cells, cell lysates (either centrifuged supernatant or non-centrifuged suspension), membrane fractions, and purified AMO protein. Briefly, N. halophila ZJB1 was cultured to the late exponential growth phase, and approximately 3 g of cells were harvested via filtration through a 0.22 μm membrane filter (Millipore). The resulting cell pellet was resuspended in 50 mL of SEC buffer (OD ≈5.0) and aliquoted into sterile 1.5 mL Eppendorf tubes (1 mL per tube). Cell disruption was carried out using an ultrasonic disruptor (Xinzhi Instrument, Zhejiang, China) under the following conditions: 80 W power output, with a 2 s pulse followed by a 4 s pause, for a total duration of 3 min. The disrupted cell suspension was either used directly as the non-centrifuged cell lysate or further processed by centrifugation. For preparation of the centrifuged extract, the lysate was centrifuged at 2000×g at 4 °C for 20 min to remove intact cells and large cellular debris. Total protein concentration was determined using a micro-UV spectrophotometer (Bio-Rad). The non-centrifuged lysate exhibited a protein concentration of approximately 22.3 mg/mL, whereas the centrifuged supernatant showed a reduced concentration of 12.1 mg/mL—approximately half that of the non-centrifuged sample. To account for this loss during centrifugation, twice the initial biomass was used in the preparation of centrifuged extracts to ensure equivalent protein loading in downstream assays.
For the membrane fractions, ~3 g of N. halophila ZJB1 cell pellet was carried to ultrasonic disruption. The resulting lysate was centrifuged at 21,500×g and 4 °C for 10 min. The supernatant was collected and then ultracentrifuged at 150,000×g and 4 °C for 1 h. After discarding the final supernatant, the membrane pellet was resuspended in 10 mL of SEC buffer. The total protein concentration of this membrane fractions was determined to be 9.5 mg/mL. Given that the AMO protein constitutes approximately 80% of the total membrane protein65, the estimated AMO protein concentration in this membrane fractions was about 7.6 mg/mL. The AMO protein was purified according to the method described above in this study, yielding a final concentration of ~3.0 mg/mL.
To investigate the factors influencing AMO activity, a series of reductants was evaluated using intact AOB cells and cell lysates. The reductants tested included duroquinone (5 mM), formate (26 mM), and NADH (10 mM), which have been widely employed as effective electron donors for measuring pMMO activity in methanotrophs28,30,66,67. In addition, the effects of several potential reductants or electron donors on the ammonia oxidation activity of intact cells and cell lysates were investigated, including ubiquinol (CoQ H , 50 μM), sodium ascorbate (Asc., 10 mM), DL-dithiothreitol (DTT, 10 mM), and tris (2-carboxyethyl) phosphine hydrochloride (TCEP, 10 mM). In reductant or electron donor assays, 1 mL of intact cell suspension or cell suspension or cell lysate was transferred to a 10 mL centrifuge tube. To this, 1 mL of SEC buffer, NH ⁺ (~5 mM), KH PO (12 mM), NaHCO (13 mM), and essential trace metals for AOB (1 mL/L) were added. A negative control without any reductant was included in each experimental series. For activity assays using membrane fractions and purified AMO protein, 1 mL of sample was transferred into 10 mL centrifuge tubes; the membrane fractions were subsequently supplemented with 1 mL of SEC buffer to achieve a final volume of 2 mL. The reaction mixtures were supplemented with CoQ H (50 μM), NH ⁺ (~5 mM), KH PO (12 mM), NaHCO (13 mM), and essential trace metals for AOB (1 mL/L). A blank control containing CoQ H but lacking membrane fractions or purified AMO protein was also included. All assays were incubated in the dark at 30 °C with shaking at 120 rpm in a temperature-controlled incubator. Aliquots were collected at predetermined time intervals to measure NH ⁺ consumption, allowing for the monitoring of ammonia oxidation activity.

Construction of mutants and enzyme activity assays
DNA fragments containing point mutations at specific amino acid residues associated with Cu and Cu were generated. Briefly, PCR amplification was carried out using the primers listed in Supplementary Table 2, and the resulting PCR products were purified. Following the cloning procedure described previously in this study, the mutated DNA fragments were initially ligated into a kanamycin resistance gene-free pJTU1278 vector to reconstruct the complete amoC gene. Subsequently, a kanamycin resistance cassette (KmR) and the amoA gene were inserted downstream through ligation. A final PCR was then performed using the primer pair amoC-F1/amoA-R1, and the amplified product was purified to yield the full-length mutated DNA fragment. The constructed fragments were introduced into the N. halophila ZJB1 wild-type strain via electroporation as described above. Transformants were selected on agar plates supplemented with 25 mg/L kanamycin, yielding six kanamycin-resistant mutant strains.
Ammonia oxidation activity of the mutant strains was assessed by monitoring NH + consumption over time. In brief, each mutant culture was adjusted to an OD of approximately 0.06 and transferred into AOB growth medium to a final volume of 10 mL, with NH + provided at a final concentration of ~2 mM along with other essential nutrients required for AOB growth. Cultures were incubated at 30 °C under shaking conditions (120 rpm). Aliquots were collected at regular intervals to determine NH + concentrations, allowing for the evaluation of ammonia oxidation activity in each mutant.

Lipidomics analysis
The lipid extraction and LC-MS/MS analysis for N. halophila ZJB1 cells and purified AMO protein were performed for three independent biological replicates, respectively. Internal standards, including TG (16:0-d9/16:0/16:0), SM (d18:1/16:0-d9), DG (16:0-d9/16:0), CE (16:0-d9), PC (16:0-d9/16:0), PE (16:0-d9/16:0), PI (16:0-d9/16:0), PS (16:0-d9/16:0) and LysoPC (16:0-d9/0:0), were added to N. halophila ZJB1 cells and AMO protein samples suspended in 1 mL of phosphate-buffered saline:methanol (1:1, v/v). The mixture was homogenized using a Precellys homogenizer (Retsch) through three cycles of 30 s at 8000 rpm, with 5 min incubations on ice between each cycle.
A methyl tert-butyl ether (MTBE)-based liquid-liquid extraction method was subsequently performed. Four milliliters of MTBE were added to each sample and vortexed vigorously. The mixtures were incubated for 1 h at room temperature on a platform shaker (Shanghai Jinsin Technology Co., Ltd.) operating at 500 rpm. Phase separation was achieved by the addition of 1 mL of water. After shaking for 10 min, the samples were centrifuged at 2000×g for 15 min. The upper organic phase from each sample was carefully aspirated using a Pasteur pipette and transferred to a fresh glass tube. The residual aqueous phase was subjected to a second extraction with 2.5 mL of MTBE/methanol/water (10:3:2.5, v/v/v). Following the same shaking and centrifugation steps as described above, the resulting organic phase was collected and combined with the initial extract. The lipid extracts were dried under a stream of nitrogen using a SpeedVac vacuum concentrator. The dried lipids were reconstituted in 200 μL of n-butanol/methanol (1:1, v/v) and transferred to autosampler vials for lipidomic profiling via LC-MS (ExionLC™ AD, SCIEX) tandem MS (QTRAP 6500+, SCIEX).
The LC-MS analytical conditions were as follows: HPLC column, Shim-pack VP-ODS C18 (pore size 5.0 μm, dimensions 2×150 mm); mobile phase, water containing 0.04% acetic acid and acetonitrile containing 0.04% acetic acid; gradient program, 100:0 (v/v) at 0 min, 5:95 (v/v) at 20.0 min, 5:95 (v/v) at 22.0 min, 95:5 (v/v) at 22.1 min, and 95:5 (v/v) at 28.0 min; flow rate, 0.25 mL min⁻¹; column temperature, 40 °C; injection volume, 5 μL. The eluate was alternately directed to an electrospray ionization triple quadrupole–linear ion trap (QTRAP)–MS or an electrospray ionization QqTOF–MS. The mass spectrometer was operated under the following conditions: ion source temperature, 500 °C; ion spray voltage, +5500 V in positive mode and −4500 V in negative mode; nebulizing gas 1 (GS1), 45 psi; auxiliary gas 2 (GS2), 55 psi; curtain gas, 35 psi. In the triple quadrupole mode, each precursor-product ion pair was monitored using optimized declustering potentials and collision energies.
All mass spectrometry data were acquired and processed using the Xcalibur 3.0 software suite. Lipid annotation and data processing were performed using Lipostar software (version 1.3.1b18; Molecular Discovery). Feature detection, noise and artifact filtering, retention time alignment, and lipid identification were conducted. Peak picking, signal smoothing and alignment algorithms were applied to resolve unique m/z and retention time pairs. A retention time window of 1–32 min was implemented during analysis. Background signals arising from blank solvents, features lacking MS/MS spectra, peaks without detectable isotopic patterns, and nonspecific noise were excluded from further analysis.
The identification and quantification of lipid molecular species were performed using LipidSearch software (version 4.1; Mitsui Knowledge Industry, Tokyo, Japan). The key processing parameters were configured as follows: target database, LIPID MAPS; precursor ion mass tolerance, 5 ppm; production mass tolerance, 5 ppm; product ion intensity threshold, 5%; m-score threshold, 1; quantitative m/z tolerance, ±5 ppm; quantitative retention time (RT) window, ±0.5 min; application of the main isomer filter and ID quality filters A, B, C, and D; adductions included +H and +NH₄⁺ in positive ion mode, and –H, +HCOO⁻, and –2H in negative ion mode. The major lipid classes selected for the search including glycerophospholipids, sphingolipids, diacylglycerols, triacylglycerols and sterols—in both positive and negative ionization modes. Identified lipids were assigned confidence scores on a 1- to 4-star scale, with higher ratings indicating greater identification certainty. Only identifications rated at 2 stars or above were retained for downstream analysis. The integrated peak areas of all identified lipids were normalized to those of their respective class-specific internal standards. For the five most abundant molecular species within each lipid class—ranked by normalized peak area ratios—identifications were manually validated and confirmed through careful inspection of MS/MS fragmentation patterns and chromatographic peak profiles.

Negative stain
4 μL purified AMO sample was applied onto the freshly plasma glow-discharged carbon grids (30 sec at 15 mA current) and stained with 2% uranyl acetate twice for 60 sec each. Images were taken with a FEI Morgagni transmission electron microscope at a magnification of 730,000× under a defocus value of ~−1.5 um.

Preparation and data acquisition of cryo-EM sample
For cryo-EM sample preparation, the Au grids (Quantifoil, R2/1, 300 mesh) coated with single-layered graphene (gifted by Jiayue Su, Tsinghua University) were used to address the issue of AMO particles failing to enter the grid holes. The grids underwent glow discharge treatment (10 mA, 10 sec) and mounted on forceps in a Mark IV Vitrobot (Thermo Fisher Scientific) at 4 °C and 100% humidity. A volume of 5 μL of the fresh AMO samples were floated on the grid for 15 sec, after which the grid was plunge-frozen in liquid ethane following blotting. Cryo-EM data of AMO was collected on a Titan Krios electron microscope operating at 300 kV, equipped with a Gatan K3 Summit direct electron detector and a GIF Quantum energy filter. The automatic software-EPU68 was used for data acquisition. Images were recorded at a pixel size of 0.668 Å under a magnification of 130,000×. The defocus value of each image was set from −1.5 to −2.0 μm during data collection. Each micrograph was dose-fractioned into 32 frames with a total dose of about 50 e–/Å2.

Image processing
The beam-induced motion of the whole micrograph was corrected by MotionCor2 1.1.069. All the following data processing procedures were carried out in cryoSPARC 4.3.270. Contrast transfer function (CTF) parameters were estimated by Patch CTF estimation for the AMO, and micrographs with poor CTF fit, as determined by Manually Curate Exposures were rejected. Particle picking was carried out using Blob Picker with diameter ranging from 8 to 20 nm. Auto-picked particles were extracted by a binning factor of 2. Following two-dimensional (2D) classification, good classes were used to train Topaz71. This dataset underwent additional rounds of 2D classification and Topaz train and extraction, repeating the process four times until no further improvements were observed. All particles from these iterations were combined, and duplicated particles were removed. A total of 382,927 particles were subjected to ab initio 3D reconstruction, followed by heterogeneous refinement to generate four EM maps. The map with AMO complex contained a total of 153,286 particles, which was subjected to two rounds of AMO heterogeneous refinement to remove the bad particles using the maps generated in the previous step as references. The best map was subsequently selected for further NU-refinement72, leading to an overall resolution of 2.36 Å as estimated by the gold-standard Fourier shell correlation at a correlation cutoff value of 0.14373. Finally, DeepEMhancer74 was employed for post-processing the final maps to facilitate model building.

Model building and refinement
The predicted AlphaFold structures of AmoA, AmoB, AmoC, AmoD, and AmoE were manually docked into the cryo-EM density map using UCSF Chimera 1.1275 and subsequently adjusted in Coot 0.8.9.176 according to the corresponding density and sequence information. Lipid molecules were selected based on lipidomic data and identifiable features, and were manually fitted into the map using Coot. Water molecules within the AMO complex, particularly those near copper centers, were modeled with reference to the previously reported pMMO structure (PDB: 7S4H) and supported by mass spectrometry data31. Additional cytoplasmic and periplasmic water molecules were placed based on the pMMO structure (PDB: 7S4H) and validated by the recently published AMO structure (PDB: 9CL6). Real-space refinement and validation of the final model were carried out using Phenix 1.14-3260,77. Structural figures were prepared with UCSF Chimera 1.1.2, ChimeraX 1.378, and PyMOL 1.8.2.1 (http://www.pymol.org).

Western blot
The western blot assays were performed following the standard procedure. Briefly, the purified AMO samples obtained from gel chromatography were resolved on a 4–20% SDS-PAGE (Yeasen Biotechnology) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked with 5% skimmed milk in TBST buffer, followed by incubating with a suitable horseradish peroxidase (HRP) labeled primary antibody (Abclonal, Cat# AE024, 1:5000) for 2 h. Visualization of the blots was achieved using an enhanced chemiluminescence (ECL) kit (Tanon), and images were captured using ChemiDoc™ MP Imaging System (Bio-Rad, CA, USA).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In their manuscript titled \"Structural insights into the catalytic mechanism of ammonia monooxygenase\", Yang and co-workers investigate the structure and mechanism of ammonia monooxygenase (AMO). Ammonia oxidation is the critical first step of the nitrification pathway witin the global nitrogen cycle, converting ammonia to hydroxylamine. AMO is an integral membrane metalloenzyme with high homology to particulate methane monooxygenase (pMMO) and is of ecological relevance, as it releases the greenhouse gas N2O, one of the main drivers of global warming, as a byproduct.\nThe authors successfully determined the cryo-EM structure of AMO from a newly isolated AOB strain, Nitrosomonas halophila ZJB1, at a resolution of 2.36 Å, yielding a complete atomic model including sidechains and – more importantly – the coordinated copper ions of the enzyme. The isolation and purification of the AOB strain and subsequently the AMO complex are detailed in the supplementary materials, including phylogenetic analysis of the isolated strain, the genetic engineering for purification, and the cryo-EM sample preparation and data processing. The data as presented seem sound and well processed, although the given initial number of particles (ca. 320,000) seems low for 6,000 micrographs, given the quality of the micrographs shown in Suppl. Fig. 2 and 3.\nLike pMMO, AMO is a cylinder-shaped homotrimeric assembly of heteropentamers. Each protomer in the complex consists of five subunits: AmoA, AmoB, AmoC, a recently discovered single transmembrane protein (AmoD), and a newly identified extramembraneous subunit (AmoE). The core is composed of AmoA, AmoB, and AmoC, with AmoB forming periplasmic β-barrel domains arranged counterclockwise as a trimer. The structure is supported by 45 transmembrane helices contributed by each subunit in each protomer. This is ihe second report on a structure of AMO, with the first, at 2.8 Å resolution, determined in a native membrane environment by the group of Amy Rosenzweig in 2024 (listed as reference 25). This work had already identified the single-helix subunit that in the present work is now designated AmoD, but could not identifythe encoding gene due to a lack of resolution for the side chains. AmoD is a single transmembrane protein positioned adjacent to AmoC, with its N-terminus facing the cytoplasm and C-terminus oriented towards the periplasm. A similarly enigmatic helical structure has been observed in particulate methane monooxygenase (pMMO), a related enzyme. Phylogenetic analysis suggests AmoD is exclusively present in the N. europaea/N. mobilis lineage and its genomic location is associated with genes involved in the tricarboxylic acid (TCA) cycle and signaling molecule synthesis, leading the authors to the interesting proposal that it plays a role in signal transduction during ammonia oxidation. Two lipid molecules were observed embedded at the interface between AmoD and AmoC.\nIn addition, the present work identifies AmoE as a further accessory subunit, comprising two small helices and two flexible loops. AmoE binds to the positively-charged surface of the N-terminal cupredoxin domain of AmoB through electrostatic interactions. It also interacts with regions of AmoA and AmoC. Its genetic neighborhood includes genes encoding components of ATPase and outer membrane beta-barrel assembly machinery (BAM) complex. This led the authors to predict a probable involvement in facilitating the assembly of the AMO complex within the cell membrane. A speculative proposal, in particular because the connection of the (inner membrane protein) AMO to the BAM machinery is not clear to this reviewer.\nThe high-resolution structure of AMO revealed a total of nine copper-binding sites, with three per protomer forming two distinct copper centers. Interestingly, the dinuclear His-site (somewhat confusingly) designated CuA in some studies on pMMO, is not present in this AMO structure due to a substitution in the AmoB sequence, suggesting less conservation. CuB is considered to be an electron transfer site in pMMO, although a very similar site in the copper enzyme LPMO clearly is catalytic. In earlier work, the nuclearity of CuB has been under debate, but more recent data on pMMO already clarified its mononucler nature with the characteristic His-brace motif, and the present data on AMO now confirms this unambiguously. The active site of AMO is suggested to be at the CuC and CuD sites that feature a rather unique coordination environment for copper by including hard oxygen ligands from side chain carboxylates or amides. In AMO, the two sites form a second copper center with two simultaneously occupied copper ligands, contrasting with individual CuC and CuD sites proposed in some pMMO structures. These are nestled within a hydrophobic channel formed by transmembrane helices from AmoA and AmoC.\nThe authors also observe density features between CuC and CuD and model these as water molecules, which seems somewhat arbitrary given the map quality shown in Fig. 2c. Noting that in the AMO reaction the source of the oxygen atom of hydroxylamine is O2 rather than H2O, it seems that this now established dinuclear site would seem to be a great candidate for O2 activation similar to classical type III copper centers, and I suggest the authors may want to comment on this aspect of the mechanism. In contrast, they propose a rod-shaped density near CuC and CuD to be hydroxylamine or other intermediates, and this does not appear to be a strong point. In the map, the density feature is elongated and somewhat unclear, and quite clearly exceeds the dimensions of hydroxylamine, H2NOH, which at this resolution should present like a diatomic molecule with a compact density feature.\nFurthermore, the authors elaborate on a water-mediated hydrogen-bonding network and suggesst that it constitutes an efficient electron transfer pathway from the periplasmic CuB to the active site CuC/CuD. Six water molecules and two key residues are involved in this pathway. Extensive lipid densities were observed within the AMO structure, their distribution and arrangement mirroring the native membrane environment, suggesting the particles were preserved in a functional state, although this structure is from an isolated enzyme rather than from a native lipid environment as in the case of the concurrently reported 2.8 Å structure of AMO from N. europaea by the Rosenzweig group (ref. 25). In a direct comparison, the authors now add the identification of AmoE and a detailed description of AmoD, defining AMO as a heteropentamer, distinct from archaeal AMO that seems to be an AmoABCXYZ heterohexamer. They furthermore resolve an occupied CuD site, defining a potential dinuclear active site that – again – might be discussed in relation to O2 activation. The higher resolution achieved (2.36 Å vs. 2.8 Å) seems to have been critical in resolving features like the presence of AmoE, the occupancy and coordination of the CuD site, and the detailed water network, thus providing a more comprehensive view of the proposed electron transfer pathway and active site.\nIn summary, this study represents a significant step forward in understanding the molecular architecture and catalytic mechanism of AMO by providing the most highly resolved and complete high-resolution cryo-EM structure. The determination of the complete heteropentameric composition, including the identification and proposed functional roles of AmoD and AmoE, adds crucial details to the understanding of this complex enzyme. The detailed mapping of copper centers, particularly the dinuclear CuC/CuD site, and the elucidation of the water-mediated electron transfer pathway provide a strong structural basis for the catalytic mechanism. Furthermore, the insights into the unique surface electrostatic potentials highlight possible mechanisms for substrate assimilation and transport. The study successfully integrates structural biology with bioinformatics and biochemical methods to provide a comprehensive view of AMO. The findings significantly enhance our understanding of this pivotal enzyme in the nitrogen cycle and its distinction from related enzymes like pMMO.\nMinor Points\nNote that page numbers at the bottom of the page in the article file are not correct after page 9. In the following I use consecutive numbering of pages in the document from page 9 onwards.\n• p.2, l.2: \"...composing five subunits.\" should be \"...composed of five subunits.\"\n• p.2, l.8: \"...cytoplasmically active site CuD...\" should be \"...cytoplasmic active site CuD...\"\n• p. 4, l. 13: \"Notably, the recent discovered single transmembrane protein...\" should be \"Notably, the recently discovered single transmembrane protein...\"\n• p.4, l.14: \"...newly identified components AmoE...\" should be \"...newly identified component AmoE...\"\n• p.9, l.8: \"Close inspection of three coppers’ coordination environment reveals...\" could be phrased more clearly, e.g., \"Close inspection of the coordination environments of the three coppers reveals...\"\n• p.10, l.10: \"...a newly identified component (NZ4318 and NZ3624) were discovered at the periphery of AMO based on the high-resolution structure at the side-chain level (Supplementary Fig. 4), assigned as AmoD and AmoE respectively.\" This sentence is confusingly phrased, as it presents two NZ numbers as a single \"newly identified component\" before assigning them separately. As I understand, NZ4318 is AmoD (the recently reported single TM protein) and NZ3624 is AmoE (the newly identified soluble protein).\n• p.12, l.4: \"...with its genomic location is associated...\" should be \"...with its genomic location associated...\"\n• p.14, l.4: \"...oxygen and electron\" should be \"...oxygen and electrons\" (plural).\n• P.15, l.2: \"In contrast their structure,\" should be \"In contrast to their structure,\"\nIn Supplementary Information:\n• p.2, l.13: \"AmoE sequence from N. halophila ZJB1 wAS used\" should be \"The AmoE […] was used\".\n• p.5, l.18: \"Peak factions were collected\" should be \"Peak fractions were collected\".\n• p.6, l.21: \"...micrograph with poor CTF fit...\" should be \"...micrographs with poor CTF fit...\"\n• p.6, l.22: \"Particles picking was carried out\" should be \"Particle picking was carried out\".\n• p.7, l.7: \"...were subjected for ab initio 3D reconstruction,\" should be \"...were subjected to ab initio 3D reconstruction,\", or “… were used for …”.\n• p.7, l.10: \"...was subjected to two rounds of AMO heterogeneous\" is incomplete; I guess “refinement” should be added after “heterogeneous”\n• p.13, l.15: \"sequences alignments\" should be \"sequence alignments\".\n• Suppl. Table 1: \"WateRs\" is a typo for \"Waters\".", "reviewer_2": "Dong & Sui et al., titled \"Structural insights into the catalytic mechanism of ammonia monooxygenase,\" reported their recent cryo-electron microscopy (cryo-EM) structure of ammonia monooxygenase (AMO), a protein complex from ammonia-oxidizing bacteria (AOB). In their research, the authors engineered the AmoA subdomain with a 3xFlag tag, co-expressed with the subunits of AmoB and AmoC, utilizing a heterogeneous vector. This construct was transformed and expressed in an AOB strain of Nitrosomonas halophila ZJB1. The resulting AmoABCDE membrane protein complexes were purified through an affinity column and subsequently collected for cryo-EM study.\nIn the cryo-EM study, the authors identified a periplasmic Cu(B) site in an AmoA subunit and a dinuclear copper (Cu(C) and Cu (D)) in an AmoC subunit. They proposed that the AMO complex facilitates ammonium oxidation through an effective electron transfer pathway from Cu(B) to the dinuclear copper site, enabling substrate oxidation. However, the study lacked direct evidence, unlike earlier studies published in the ref. 36 and 38, unequivocally demonstrating substrate binding, specifically ammonium or hydroxylamine, to the proposed dinuclear active site.\nThe copper sites of Cu(B), Cu(C), and Cu(D) have already been identified in various pMMO X-ray diffraction and cryo-EM structures. A recent cryoEM structure of AMO mentioned in the ref. 25 also revealed similar copper configurations for the Cu(B) and Cu(C) sites. Additionally, this study identified a corresponding Cu(D) site composed of Asn 207, His 211, and His 225, which may act as potential coordination ligands. Overall, the research progress achieved in this study is incremental.\nThe purified AMO complex is a heterogeneously expressed variant with a 3xFlag tag. It is necessary to perform a series of tests and characterizations related to AMO activity, including mechanistic and spectroscopic analyses, to verify that the obtained recombinant protein complex variants demonstrate AMO activity with high performance. The study also speculates on the potential structural differences between pMMO and AMO to provide mechanistic insights, although there is currently no strong evidence for these speculations. A more quantitative investigation is needed to achieve a fundamental understanding of how AMO facilitates biological ammonia oxidation at the molecular level. The results presented in this study are still preliminary, and this reviewer does not recommend the publication of the work in its present format.", "reviewer_3": "Yang et al., report a structure of an ammonia monooxygenase enzyme (AMO complex) from the ammonia-oxidising bacterium (AOB) Nitrosomonas halophila, and from a new strain that the authors isolated themselves from mangrove sediments. They have engineered a tagged version of the subunit Amo-A that they have used to isolate the full complex that they have analysed by cryo-EM. At the end of 2024, another group also obtained the structure of an AMO complex of the same family, Nitrosomonas europaea (Tucci et al., 2024). This study by Yang et al. is only slightly incremental compared to Tucci et al, as they manage to find the identity of the long transmembrane helix AMOD and to visualise a new AMOE short helical protein. The cryo-EM maps look nice, but the manuscript falls short of explanations and insights all through the text. The introduction could be better and more readable by explicitly explaining some concepts the are mentioned, but are not known by readers that are not in the field of mono oxygenates. As an example in line 6 the authors write “consisting 6 of three components and controversial copper-binding sites”, they could explain what this controversy is about. Does it have to do with the fact that in some of the pMMO structures Zn instead of Cu is found in the metal centers?\nRegarding this, we have a major concern, how do the authors know that they have Cu rather than Zn bound in their structure? By EM density analysis they cannot be certain about it. We suggest to perform some sort of X-ray spectroscopy to identify the element. The authors mention in line 14 that O has been established to be a Cu dependent oxygenase, but there is no reference. This lack of citations is spread all over the text. It is hard to follow for someone that doesn’t know the specific literature.\nThe main text, results and discussion, is a description of the structure, but lacks completely functional analysis and argumentation about the role of certain observations. We consider and understand that in vitro biochemical characterisation of such en enzyme is very difficult, but some level of in-cell functional study should be possible and would make the paper more valuable. The authors could manipulate the bacteria strain to make a AMO-A tagged version. Is it not possible to include mutations in the various metal centres and then check the level N2O formation?\nThis would be really necessary to prove that the water path is important for e- transfer (as the authors state) and could be used to prove the physiological relevance of the double-copper presence at the Cu-D and Cu-C site.\nWithout any other type of assay, the structure analysis remains quite sterile, unfortunately.\nThe analysis of the lipids and their distribution is completely relegated to the supplementary materials, not attempt to identify the type of lipids has been done (e.g. mass spec?) and the lipids are not built in the deposited pdb. Why? We noticed that also the other group’s structure (Tucci et al. pdb-9cl6) doesn’t contain the lipids. On the other hand, very many water molecules were built in the maps, which is a bit surprising at this resolution. For some water molecules there is no density and for other water molecules the density is ambiguous and might also represent noise. The high-resolution noise and intrinsic motion of the outer side of the cryo-EM maps, makes it difficult to believe that all those H2O molecule can be built in the periphery of the structure. In the supplementary part about the methods the author are completely missing the description about model building. This should be corrected. We wonder whether the model was build starting from previous homologous structures that already included the waters. In any case the procedure that was followed to build the model should be described.\nGoing through the text:\n- Page 3 Line 13: which are the two genes downstream of amoCAB that were considered accessory components of AMO in AOB?\n- Page 4, line 6: what do the authors mean by “components”?\n- As said before, mutagenesis of the key residues identified would be really useful to gain mechanistic information\n- Page 8 Line 8an almost 4Å bond is labelled as H-bond this is a bit too far away\n- Page 8 Line 16-17 The authors compare one TM helix in their complex with the correspondent one in pMMO. It would be useful to have a sequence alignment to see how conserved this helix is and if there are specific aa that would explain the kink of the helix.\n- Line 8-12 page 9 the manuscript reads:\nClose inspection of three coppers’ coordination environment reveals that a relatively weak hydrogen bond (Fig. 2b, e and f, red stars) consistently accompanies these coppers, suggesting their potential role in facilitating electron transfer during substrate oxidation, as previously reported, beyond merely stabilizing the copper site44-46.\nHow does the hydrogen bond network facilitate electron transfer? Could you please give a reference for such mechanism? Could the electrons not tunnel directly from metal center to metal center given that the distance is less than 20 Å? Do the authors maybe instead mean proton transfer to the active site?\n- Page 9, line 15 the rod-shaped density could also represent noise instead of hydroxylamine\n- Page 10 Line 1 it is confusing tha the bis-His site in introduced all of a sudden with no context, nor it was given in the introduction and with references lacking.\n- Page 10 Line 14-16 . Notably, AmoD is positioned adjacent to AmoC with its N-terminal facing the cytoplasmic side and C-terminal oriented towards the periplasmic side (Fig. 1 and 3a).\nWhy notably? What is the functional implication of this observation?\nAnd again on then following page The first alkyl chain of this lipid interacts with AmoD via Trp37 and penetrates into the hydrophobic core of AmoD and AmoC, while the second alkyl chain of this lipid anchors onto the surface of the TM1 of AmoC and stretches into the lipid bilayer of the cell membrane (Fig. 3c).\nWhat are the physiological implications of such observation?\n- Based on the bioinformatics analysis a possible role is assigned to AMO-E, but it sis unclear. How is ammonia oxidation related to the TCA cycle ? This is most likely due to our ignorance, but it would be better of the authors would explain the correlation between the two enzymatic processes.\n- It is unclear why the positive electrostatic charge distribution at the membrane levels should play a very important role.\n- The conclusions seem a bit misleading and not completely fair. First the assembly is described as unexpected while it cannot be the case view that the other AMO structure from Tucci et al, was already available and then they conclude with a comment about the negative aspects of the structure from the other lab instead of focusing on what could have been novel in their presented structure.\nTaking all this into account, we suggest that a substantial effort is put by the authors in some assays that would highlight the physiological importance of their structural observations. Moreover, the text is missing crucial references in several places (for example Page 7, line 16, a reference to the comparison AMO structure is needed). Such references should be put all throughout the text.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Thank you for your letter and for the reviewers’ insightful comments on our manuscript\n(Research Article, No. NCOMMS-25-30401). We greatly appreciate the time and effort\ndedicated to providing constructive feedback, which has been invaluable in improving\nour paper.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn their manuscript titled \"Structural insights into the catalytic mechanism of ammonia\nmonooxygenase\", Yang and co-workers investigate the structure and mechanism of\nammonia monooxygenase (AMO). Ammonia oxidation is the critical first step of the\nnitrification pathway within the global nitrogen cycle, converting ammonia to\nhydroxylamine. AMO is an integral membrane metalloenzyme with high homology to\nparticulate methane monooxygenase (pMMO) and is of ecological relevance, as it\nreleases the greenhouse gas N2O, one of the main drivers of global warming, as a\nbyproduct.\nThe authors successfully determined the cryo-EM structure of AMO from a newly\nisolated AOB strain, Nitrosomonas halophila ZJB1, at a resolution of 2.36 Å, yielding\na complete atomic model including sidechains and – more importantly – the\ncoordinated copper ions of the enzyme. The isolation and purification of the AOB strain\nand subsequently the AMO complex are detailed in the supplementary materials,\nincluding phylogenetic analysis of the isolated strain, the genetic engineering for\npurification, and the cryo-EM sample preparation and data processing. The data as\npresented seem sound and well processed, although the given initial number of particles\n(ca. 320,000) seems low for 6,000 micrographs, given the quality of the micrographs\nshown in Suppl. Fig. 2 and 3.\nReply: We sincerely thank the reviewer for the positive assessment of our work and\ninsightful comments. The successful determination of the high-resolution structure of\nthe AMO complex in this study relied critically on the establishment of an effective in\nvitro isolation and purification workflow using genetically engineered endogenous\ntagging. We acknowledge that the current protein yield remains relatively limited,\nwhich is partly attributable to the presence of multiple copies of AMO-encoding genes\nin ammonia-oxidizing bacteria. Specifically, from approximately 120 liters of AOB\nculture, we were able to obtain about 20 µL of highly concentrated, quality AMO\ncomplex. As shown in the revised Supplementary Fig. 4, the particle concentration in\nthe cryo-EM samples was indeed relatively low, with only dozens of particles per\nmicrograph, ultimately yielding an initial particle set of approximately 380,000.\nDespite this constraint, the genetic manipulation system and purification strategy we\ndeveloped provide a solid foundation for future structural and functional studies of the\nAMO complex. We are confident that further optimization will enhance the yield and\nfacilitate more extensive investigations in this field.\nLike pMMO, AMO is a cylinder-shaped homotrimeric assembly of heteropentamers.\nEach protomer in the complex consists of five subunits: AmoA, AmoB, AmoC, a\nrecently discovered single transmembrane protein (AmoD), and a newly identified\nextramembraneous subunit (AmoE). The core is composed of AmoA, AmoB, and\nAmoC, with AmoB forming periplasmic β-barrel domains arranged counterclockwise\nas a trimer. The structure is supported by 45 transmembrane helices contributed by each\nsubunit in each protomer. This is the second report on a structure of AMO, with the first,\nat 2.8 Å resolution, determined in a native membrane environment by the group of Amy\nRosenzweig in 2024 (listed as reference 25). This work had already identified the\nsingle-helix subunit that in the present work is now designated AmoD, but could not\nidentify the encoding gene due to a lack of resolution for the side chains. AmoD is a\nsingle transmembrane protein positioned adjacent to AmoC, with its N-terminus facing\nthe cytoplasm and C-terminus oriented towards the periplasm. A similarly enigmatic\nhelical structure has been observed in particulate methane monooxygenase (pMMO), a\nrelated enzyme. Phylogenetic analysis suggests AmoD is exclusively present in the N.\neuropaea/N. mobilis lineage and its genomic location is associated with genes involved\nin the tricarboxylic acid (TCA) cycle and signaling molecule synthesis, leading the\nauthors to the interesting proposal that it plays a role in signal transduction during\nammonia oxidation. Two lipid molecules were observed embedded at the interface\nbetween AmoD and AmoC.\nIn addition, the present work identifies AmoE as a further accessory subunit,\ncomprising two small helices and two flexible loops. AmoE binds to the positively-\ncharged surface of the N-terminal cupredoxin domain of AmoB through electrostatic\ninteractions. It also interacts with regions of AmoA and AmoC. Its genetic\nneighborhood includes genes encoding components of ATPase and outer membrane\nbeta-barrel assembly machinery (BAM) complex. This led the authors to predict a\nprobable involvement in facilitating the assembly of the AMO complex within the cell\nmembrane. A speculative proposal, in particular because the connection of the (inner\nmembrane protein) AMO to the BAM machinery is not clear to this reviewer.\nReply: We sincerely thank the reviewer for raising this important point regarding the\nfunctional connection between AmoE and the BAM complex.\nThe structure of AMO determined in this study reveals that the newly identified AmoE\nsubunit is located on the periplasmic side of the complex. While the genetic\nneighborhood of amoE includes genes encoding components of the ATPase and the\nouter membrane beta-barrel assembly machinery (BAM) complex, we agree that the\nfunctional implication of this genomic association remains unclear.\nA recent cryo-electron tomography (cryo-ET) study of AOA, combined with cross-\nlinking mass spectrometry analysis of archaeal membranes, suggested a potential\nspatial proximity between the extracellular S-layer and the AMO complex, which may\nfacilitate substrate uptake1,2. Although this does not directly clarify the role of AmoE,\nit hints at possible functional interactions between ammonia-oxidizing enzymes and\nouter membrane-associated components.\nIn light of the reviewer’s comment, we have toned down our interpretation and revised\nthe manuscript to reflect a more cautious hypothesis. We now propose that AmoE might\nserve as a periplasmic adaptor, potentially facilitating structural or functional\ncommunication across the periplasm—possibly helping to coordinate electron transfer,\nmetabolite channeling, or complex stability—without implying a direct role for BAM\nin AMO biogenesis.\nThe revised statement now reads:\n“In contrast, the genetic neighborhood of amoE encompasses genes encoding\ncomponents of the ATPase and outer membrane beta-barrel assembly machinery\ncomplex (Fig. 3h and Supplementary Fig. 10a and c), suggesting a potential, though\nstill speculative, functional relationship. Given its periplasmic localization, we\nhypothesize that AmoE may help coordinate interactions between the inner membrane-\nembedded AMO complex and outer membrane-associated components, possibly\ncontributing to regulatory or substrate delivery processes. This is consistent with the\npreviously predicted interaction between the AMO and the S-layer outside the\nmembrane in archaea. However, further experimental validation is required to elucidate\nits precise role.”\nWe thank the reviewer again for prompting this clarification, which has helped us\nimprove the accuracy of our discussion.\nThe high-resolution structure of AMO revealed a total of nine copper-binding sites,\nwith three per protomer forming two distinct copper centers. Interestingly, the dinuclear\nHis-site (somewhat confusingly) designated CuA in some studies on pMMO, is not\npresent in this AMO structure due to a substitution in the AmoB sequence, suggesting\nless conservation. CuB is considered to be an electron transfer site in pMMO, although\na very similar site in the copper enzyme LPMO clearly is catalytic. In earlier work, the\nnuclearity of CuB has been under debate, but more recent data on pMMO already\nclarified its mononucler nature with the characteristic His-brace motif, and the present\ndata on AMO now confirms this unambiguously. The active site of AMO is suggested\nto be at the CuC and CuD sites that feature a rather unique coordination environment\nfor copper by including hard oxygen ligands from side chain carboxylates or amides.\nIn AMO, the two sites form a second copper center with two simultaneously occupied\ncopper ligands, contrasting with individual CuC and CuD sites proposed in some\npMMO structures. These are nestled within a hydrophobic channel formed by\ntransmembrane helices from AmoA and AmoC.\nThe authors also observe density features between CuC and CuD and model these as\nwater molecules, which seems somewhat arbitrary given the map quality shown in Fig.\n2c. Noting that in the AMO reaction the source of the oxygen atom of hydroxylamine\nis O2 rather than H2O, it seems that this now established dinuclear site would seem to\nbe a great candidate for O2 activation similar to classical type III copper centers, and I\nsuggest the authors may want to comment on this aspect of the mechanism. In contrast,\nthey propose a rod-shaped density near CuC and CuD to be hydroxylamine or other\nintermediates, and this does not appear to be a strong point. In the map, the density\nfeature is elongated and somewhat unclear, and quite clearly exceeds the dimensions of\nhydroxylamine, H2NOH, which at this resolution should present like a diatomic\nmolecule with a compact density feature.\nReply: We sincerely appreciate the reviewer’s insightful comments. The assignment of\nwater molecules within the electron density between the Cu and Cu sites was guided\nC D\nby structural comparisons with previously reported pMMO structures (PDB IDs: 3RGB\nand 7S4H), which exhibit water molecules localized near the Cu and Cu sites,\nC D\nrespectively. This interpretation is further corroborated by a recently published AMO\nstructure (PDB ID: 9CL6), which also features water molecules in proximity to the Cu\nC\nsite (Response Figure 1).\nThe simultaneous occupancy of both Cu and Cu sites observed in this study bears\nC D\nresemblance to classical type III copper centers; however, notable differences exist in\ntheir coordination environments. In classical type III copper centers, three histidine\nresidues serve as coordinating ligands, whereas in AMO, each copper site is coordinated\nby two histidine residues and two negatively charged residues. Moreover, the Cu–Cu\nbond length in classical type III copper centers typically ranges from 3.6 to 4.6 Å3,\nwhich is significantly shorter than the 7.9 Å distance observed between Cu and Cu\nC D\nin our study (Response Figure 1a-c).\nBased on the catalytic mechanism of classical type III copper centers, O could be\n2\nconsidered a plausible candidate for the observed density between Cu and Cu .\nC D\nHowever, the typical Cu–O bond length in such systems (1.8–2.2 Å) is notably shorter\nthan the distances measured between the observed density and Cu (2.6 Å) or Cu (3.6\nC D\nÅ), respectively (Response Figure 1d and e). Furthermore, water molecules have been\nreported to bridge the two copper ions in the resting met-form of type III copper centers4,\nas modeled in the pMMO structures (PDB IDs: 3RGB and 7S4H). Therefore, we\npropose that the observed densities most likely correspond to water molecules. That\nsaid, we currently lack conclusive evidence to definitively exclude the presence of\nmolecular oxygen.\nIn response to the reviewer’s suggestion, we have revised the relevant sections and\nincorporated a discussion on classical type III copper centers as follows:\n“Between the Cu and Cu ligands, we observe two distinct densities (Fig. 2c). While\nC D\nthese are tentatively modeled as water molecules based on precedents in related\nmetalloenzyme structures, we cannot exclude the possibility that they represent oxygen\nspecies derived from dioactivation. The dinuclear nature of this center invites\ncomparison to type III copper sites involved in O binding and activation; however, the\n2\nlonger metal–metal distance and atypical coordination geometry suggest a distinct\nmechanism in AMO”\nThis portion of the results has been integrated into the revised Supplementary Fig. 8.\nResponse Figure 1. Simultaneously co-occupied Cu -Cu in AMO suggest a\nC D\ndistinct catalytic mechanism. a-e Comparisons of coordination environments of\ncoppers in Cu and Cu site from pMMO (a, b), Cu site from recently reported AMO\nC D C\n(c), the classical type III copper center from Juglans regia Tyrosinase (jrTYR) (d) and\nthe dicopper center from AMO resolved in this study (e). Copper ligands (Cu), oxygen\n(O) and waters (W) are shown as cyan, red and green spheres respectively.\nAdditionally, due to the limited resolution of the structural data, the rod-shaped density\nobserved between Cu and Cu remains ambiguous in terms of definitive assignment.\nC D\nAccordingly, we have revised the corresponding description in the revised manuscript\nas follows:\n“Interestingly, an elongated, rod-shaped density is observed in the hydrophobic channel\nadjacent to the Cu –Cu center (Fig. 2c). While its identity remains uncertain, its\nC D\nlocation suggests a potential role in substrate access or product release. Further studies\nat higher resolution or under different catalytic conditions will be necessary to elucidate\nthe nature of this feature.”\nFurthermore, the authors elaborate on a water-mediated hydrogen-bonding network and\nsuggesst that it constitutes an efficient electron transfer pathway from the periplasmic\nCuB to the active site CuC/CuD. Six water molecules and two key residues are involved\nin this pathway. Extensive lipid densities were observed within the AMO structure, their\ndistribution and arrangement mirroring the native membrane environment, suggesting\nthe particles were preserved in a functional state, although this structure is from an\nisolated enzyme rather than from a native lipid environment as in the case of the\nconcurrently reported 2.8 Å structure of AMO from N. europaea by the Rosenzweig\ngroup (ref. 25). In a direct comparison, the authors now add the identification of AmoE\nand a detailed description of AmoD, defining AMO as a heteropentamer, distinct from\narchaeal AMO that seems to be an AmoABCXYZ heterohexamer. They furthermore\nresolve an occupied CuD site, defining a potential dinuclear active site that – again –\nmight be discussed in relation to O2 activation. The higher resolution achieved (2.36 Å\nvs. 2.8 Å) seems to have been critical in resolving features like the presence of AmoE,\nthe occupancy and coordination of the CuD site, and the detailed water network, thus\nproviding a more comprehensive view of the proposed electron transfer pathway and\nactive site.\nIn summary, this study represents a significant step forward in understanding the\nmolecular architecture and catalytic mechanism of AMO by providing the most highly\nresolved and complete high-resolution cryo-EM structure. The determination of the\ncomplete heteropentameric composition, including the identification and proposed\nfunctional roles of AmoD and AmoE, adds crucial details to the understanding of this\ncomplex enzyme. The detailed mapping of copper centers, particularly the dinuclear\nCuC/CuD site, and the elucidation of the water-mediated electron transfer pathway\nprovide a strong structural basis for the catalytic mechanism. Furthermore, the insights\ninto the unique surface electrostatic potentials highlight possible mechanisms for\nsubstrate assimilation and transport. The study successfully integrates structural\nbiology with bioinformatics and biochemical methods to provide a comprehensive view\nof AMO. The findings significantly enhance our understanding of this pivotal enzyme\nin the nitrogen cycle and its distinction from related enzymes like pMMO.\nMinor Points\nNote that page numbers at the bottom of the page in the article file are not correct after\npage 9. In the following I use consecutive numbering of pages in the document from\npage 9 onwards.\nReply: We thank the reviewer for their careful reading and for identifying this error. We\nhave thoroughly checked the page numbering throughout the entire document to ensure\nconsistency and accuracy.\n• p.2, l.2: \"...composing five subunits.\" should be \"...composed of five subunits.\"\nReply: We thank the reviewer for pointing out this. The correction has been made in the\nrevised manuscript.\n• p.2, l.8: \"...cytoplasmically active site CuD...\" should be \"...cytoplasmic active site\nCuD...\"\nReply: We thank the reviewer for pointing out this. Considering that the exact location\nof Cu ligand is within the inner membrane, we have corrected it in the revised version\nD\nas follows:\n“Three modeled coppers, along with an adjacent water-mediated hydrogen-bond\nnetwork, may facilitate an efficient proton transfer pathway from the periplasmic Cu\nB\nto the active site Cu within the inner membrane, where Cu and Cu act in concert to\nD C D\ncatalyze substrate reaction.”\n• p. 4, l. 13: \"Notably, the recent discovered single transmembrane protein...\" should be\n\"Notably, the recently discovered single transmembrane protein...\"\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion.\n• p.4, l.14: \"...newly identified components AmoE...\" should be \"...newly identified\ncomponent AmoE...\"\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion.\n• p.9, l.8: \"Close inspection of three coppers’ coordination environment reveals...\" could\nbe phrased more clearly, e.g., \"Close inspection of the coordination environments of the\nthree coppers reveals...\"\nReply: We thank the reviewer for pointing out this. We have rewritten it in the revised\nversion as follows:\n“Close inspection of the coordination environments of the three coppers reveals the\nconsistent presence of a relatively weak hydrogen bond associated with these sites (Fig.\n2b, d and e, red stars), suggesting their potential role in facilitating proton transfer\nduring substrate oxidation, beyond merely stabilizing the copper sites.”\n• p.10, l.10: \"...a newly identified component (NZ4318 and NZ3624) were discovered\nat the periphery of AMO based on the high-resolution structure at the side-chain level\n(Supplementary Fig. 4), assigned as AmoD and AmoE respectively.\" This sentence is\nconfusingly phrased, as it presents two NZ numbers as a single \"newly identified\ncomponent\" before assigning them separately. As I understand, NZ4318 is AmoD (the\nrecently reported single TM protein) and NZ3624 is AmoE (the newly identified soluble\nprotein).\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion as follows:\n“Beyond the aforementioned three core subunits, the structure of AMO resolved in this\nstudy reveals a recently reported single transmembrane protein (identified as NZ4318\nin this study) and a newly identified component (NZ3624) at the periphery of AMO\nbased on the high-resolution structure at the side-chain level (Supplementary Fig. 5),\nassigned as AmoD and AmoE, respectively.”\n• p.12, l.4: \"...with its genomic location is associated...\" should be \"...with its genomic\nlocation associated...\"\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion.\n• p.14, l.4: \"...oxygen and electron\" should be \"...oxygen and electrons\" (plural).\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion.\n• P.15, l.2: \"In contrast their structure,\" should be \"In contrast to their structure,\"\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion and reorganized the “Conclusion” section.\nIn Supplementary Information:\n• p.2, l.13: \"AmoE sequence from N. halophila ZJB1 wAS used\" should be \"The AmoE\n[…] was used\".\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion.\n• p.5, l.18: \"Peak factions were collected\" should be \"Peak fractions were collected\".\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion.\n• p.6, l.21: \"...micrograph with poor CTF fit...\" should be \"...micrographs with poor\nCTF fit...\"\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion.\n• p.6, l.22: \"Particles picking was carried out\" should be \"Particle picking was carried\nout\".\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion.\n• p.7, l.7: \"...were subjected for ab initio 3D reconstruction,\" should be \"...were\nsubjected to ab initio 3D reconstruction,\", or “… were used for …”.\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion.\n• p.7, l.10: \"...was subjected to two rounds of AMO heterogeneous\" is incomplete; I\nguess “refinement” should be added after “heterogeneous”\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion.\n• p.13, l.15: \"sequences alignments\" should be \"sequence alignments\".\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion.\n• Suppl. Table 1: \"WateRs\" is a typo for \"Waters\".\nReply: We thank the reviewer for pointing out this. We have corrected it in the revised\nversion.\nReviewer #2 (Remarks to the Author):\nDong & Sui et al., titled \"Structural insights into the catalytic mechanism of ammonia\nmonooxygenase,\" reported their recent cryo-electron microscopy (cryo-EM) structure\nof ammonia monooxygenase (AMO), a protein complex from ammonia-oxidizing\nbacteria (AOB). In their research, the authors engineered the AmoA subdomain with a\n3xFlag tag, co-expressed with the subunits of AmoB and AmoC, utilizing a\nheterogeneous vector. This construct was transformed and expressed in an AOB strain\nof Nitrosomonas halophila ZJB1. The resulting AmoABCDE membrane protein\ncomplexes were purified through an affinity column and subsequently collected for\ncryo-EM study.\nIn the cryo-EM study, the authors identified a periplasmic Cu(B) site in an AmoA\nsubunit and a dinuclear copper (Cu(C) and Cu (D)) in an AmoC subunit. They proposed\nthat the AMO complex facilitates ammonium oxidation through an effective electron\ntransfer pathway from Cu(B) to the dinuclear copper site, enabling substrate oxidation.\nHowever, the study lacked direct evidence, unlike earlier studies published in the ref.\n36 and 38, unequivocally demonstrating substrate binding, specifically ammonium or\nhydroxylamine, to the proposed dinuclear active site.\nReply: We thank the reviewer for their insightful comments and for highlighting the\nimportance of direct structural evidence for substrate binding to fully validate the\nproposed catalytic mechanism. We agree that observing ammonium or hydroxylamine\nbound at the dinuclear copper site would significantly strengthen our model.\nHowever, resolving a substrate-bound structure, particularly those involving small-\nmolecule ligands such as ammonia and hydroxylamine, to the resolution required for\nunambiguous identification of these molecules (~ 2.0 Å), remains technically\nchallenging given the current protein yield. Our purification protocol yields\napproximately 20 µL of purified AMO complex from 120 liters of Nitrosomonas\nhalophila culture—a process requiring over three weeks. Scaling this production to\nobtain the quantity and quality of sample necessary for near-atomic resolution is\ncurrently not feasible.\nInstead, to functionally probe the active site, we performed site-directed mutagenesis\nof residues coordinating the Cu and Cu centers. Most single-copy mutants showed a\nC D\nnoticeable decline (~30%) in ammonia oxidation activity (Response Figure 2a),\nsupporting the functional relevance of these sites given that the presence of three copies\nof amo operon. These effects of mutations are compare to that of the deletion of single\nAMO subunits (Response Figure 2b and c)5, suggesting an essential role of these key\nresidues in ammonia oxidation. While this does not replace direct structural evidence,\nit provides important complementary functional data. This portion of the results has\nbeen integrated into the revised Figure 2g.\nResponse Figure 2. Determination of the enzymatic activity rates in AMO single-\nmutant strains. This figure illustrates the alterations in enzymatic activity resulting\nfrom mutations in key residues involved in coordinating Cu and Cu within AMO, as\nC D\nobserved in this study (a), in comparison to previously reported data for deletions of\nAmoA1 (■) and AmoA2 (▲) (b), and AmoB1 (■) and AmoB2 (▲) (c). Wild-type\nactivity is indicated by (●)5.\nThe central advance of our work is the determination of the AMO structure, which\nrevises the subunit composition (to include AmoD and AmoE), reveals a\nsimultaneously occupied dinuclear copper center (Cu –Cu ), demonstrates a unique\nC D\nsurface electrostatic potential and proposes a proton transfer pathway. These findings\noffer a foundational structural framework for understanding AMO function. We have\nexplicitly noted in the revised manuscript that the assignment of the dinuclear site as\nthe substrate-binding center, while strongly supported by structural analogy and\nmutagenesis, awaits direct experimental confirmation in future studies.\nWe are committed to addressing this important question in future work through\ncontinued optimization of protein production and cryo-EM methodologies.\nThe copper sites of Cu(B), Cu(C), and Cu(D) have already been identified in various\npMMO X-ray diffraction and cryo-EM structures. A recent cryoEM structure of AMO\nmentioned in the ref. 25 also revealed similar copper configurations for the Cu(B) and\nCu(C) sites. Additionally, this study identified a corresponding Cu(D) site composed of\nAsn 207, His 211, and His 225, which may act as potential coordination ligands. Overall,\nthe research progress achieved in this study is incremental.\nReply: We thank the reviewer for their comment, which allows us to clarify the key\nadvance presented in our work. We agree that copper sites such as Cu , Cu , and Cu\nB C D\nhave been individually identified in previous structural studies of pMMO and AMO.\nHowever, our structure provides the first direct evidence for the simultaneous\noccupancy of both the Cu and Cu sites, forming a dicopper center within a single,\nC D\nfunctional unit. Recent quantum mechanics/molecular mechanics (QM/MM)\nsimulations have demonstrated that both Cu and Cu sites tend to maintain the Cu(I)\nC D\nstate, highlighting the potential relevance of a dicopper state; however, this state had\nnot been structurally resolved6. While the recent AMO structure (original ref. 25)\nidentified a Cu site, it did not reveal clear electron density for a dicopper center\nC\ncontaining both metal ligands, and the previously proposed active Cu site was not\nD\nobserved.\nThis finding of a dicopper center provides a fundamental structural basis for\nunderstanding the diversity of the AMO active site and the complexity of its catalytic\nmechanism. In addition, it shifts the paradigm from the long-debated question of\n“which single copper site is active” to investigating “how the dicopper center operates”,\nthereby opening new avenues for mechanistic studies and bio-inspired catalyst design.\nThe purified AMO complex is a heterogeneously expressed variant with a 3xFlag tag.\nIt is necessary to perform a series of tests and characterizations related to AMO activity,\nincluding mechanistic and spectroscopic analyses, to verify that the obtained\nrecombinant protein complex variants demonstrate AMO activity with high\nperformance. The study also speculates on the potential structural differences between\npMMO and AMO to provide mechanistic insights, although there is currently no strong\nevidence for these speculations. A more quantitative investigation is needed to achieve\na fundamental understanding of how AMO facilitates biological ammonia oxidation at\nthe molecular level. The results presented in this study are still preliminary, and this\nreviewer does not recommend the publication of the work in its present format.\nReply: We sincerely thank the reviewer for their thorough evaluation and constructive\ncomments, which have been invaluable in improving our manuscript. We have\nconducted additional experiments and analyses to address each of the concerns raised,\nas detailed below:\n1. Expression and purification of the AMO complex\nWe would like to clarify that the AMO complex was not produced via heterologous\noverexpression. Instead, a suicide plasmid was used to genomically integrate a 3×Flag\ntag at the C-terminus of AmoA, ensuring that the tagged complex is expressed under\nnative regulatory elements. This approach maintains endogenous expression levels and\ncomplex integrity, and the resulting purified complex is structurally and\ncompositionally representative of the native AMO. This similarity can also be\ncorroborated by comparison with the overall structure of another recently published\nAMO structure in native membrane7.\n2. X-ray photoelectron spectroscopy (XPS) analysis confirmed the presence of\ncopper in AMO\nAlthough previous studies have reported that AMO is copper-dependent and the\nrecently reported AMO structure has included copper ions in the protein model, direct\nexperimental evidence confirming the presence of copper ions has remained lacking.\nTo address this issue, we performed XPS experiments to identify the metal composition\nof the purified AMO sample. The XPS results showed a weak but detectable copper\nsignal, with no indication of zinc (Response Figure 3). The low-intensity copper signal\nmay be attributed to the sampling depth of XPS (typically limited to 5-10 nm), the\ncopper ligands are deeply embedded within AMO and relatively low abundance of\ncopper in AMO (3 copper ligands per AMO protomer). Although the current\nexperimental technique and sample require further optimization, our results provide\ndirect evidence for the identification of copper in AMO.\nResponse Figure 3. XPS analysis of the metal composition in AMO. The core-level\nspectra of Cu 2p (a) and Zn 2p (b) are displayed, with characteristic peaks labeled as\nCu 2p and Cu 2p , corresponding to distinct electronic states of copper. No\n1/2 3/2\ndetectable signal from zinc was observed.\n3. Activity assays and functional characterization\nwe conducted systematic activity assays using intact AOB cells and cell lysate (non-\ncentrifugal) followed by brief sonication. Initial tests using electron donors commonly\nemployed for pMMO, including NADH, duroquinol and formate, did not yield\ndetectable AMO activity (Response Figure 4). To identify a suitable electron donor for\nAMO, we screened a panel of potential compounds and found that ubiquinol (CoQ H )\n10 2\nsignificantly enhanced ammonia oxidation activity (Response Figure 5) and was\ntherefore used in all subsequent assays.\nResponse Figure 4. The electron donors used to assess the enzymatic activity of\npMMO are unsuitable for application with AMO.\nResponse Figure 5. Screening of potential electron donors for AMO reveals that\nCoQ H may be the natural electron donor for AMO.\n10 2\nWe then measured AMO activity across progressively purified cellular fractions, each\nsupplemented with CoQ H . This gradual loss of activity is consistent with the\n10 2\nbehavior observed in membrane-bound pMMO or pMMO with native lipids8 (Response\nFigure 6a), indicating that the decline is not an artifact of the 3×Flag tag or the\npurification procedure, but rather results from the disruption of native membrane\nintegrity and the loss of essential redox cofactors during isolation. Importantly, the\nammonia oxidation activity of the purified AMO sample was compared with that of the\nmembrane-bound form, indicating that the purified AMO retained substantial\nenzymatic activity (Response Figure 6b).\nThis portion of the results has been integrated into the revised Supplementary Fig. 3.\nResponse Figure 6. Determination of ammonia oxidation activity of AMO. a\nChanges in AMO enzyme activity across different cell fractions suggest efficient\nammonia oxidation relies on factors beyond CoQ H alone. b Comparison of ammonia\n10 2\noxidation activity of AMO in membrane and purified form. Ammonia oxidation activity\nwas determined based on the rate of NH ⁺ consumption over a 6-hour period.\n4\n4. Quantitative molecular investigations\nTo probe the molecular mechanism of ammonia oxidation, we used our genetic system\nto introduce point mutations into key residues coordinating the Cu and Cu centers.\nC D\nWe successfully generated strains with single-copy mutations in H140A, H153A,\nD146A, N207A, E218A, and H211A. Attempts to generate D136A and H225A mutants\nwere unsuccessful, likely due to severe functional impairments caused by the two\nmutations. Since the host genome contains three copies of the amo operon, mutation of\none copy resulted in an average reduction in activity to approximately 30% of wild-\ntype levels (Response Figure 2a). These effects of mutations are compare to that of the\ndeletion of pMMO subunits5 (Response Figure 2b and c), supporting the essential role\nof these residues in catalysis and providing direct molecular evidence for the proposed\nmechanism of AMO.\nThis portion of the results has been integrated into the revised Figure 2g.\nResponse Figure 2. Determination of the enzymatic activity rates in AMO single-\nmutant strains. This figure illustrates the alterations in enzymatic activity resulting\nfrom mutations in key residues involved in coordinating Cu and Cu within AMO, as\nC D\nobserved in this study (a), in comparison to previously reported data for deletions of\nAmoA1 (■) and AmoA2 (▲) (b), and AmoB1 (■) and AmoB2 (▲) (c). Wild-type\nactivity is indicated by (●)5.\nWe are confident that these revisions have significantly strengthened the study and\nprovided a more quantitative and mechanistic foundation for understanding AMO\nfunction. We thank the reviewer again for their thoughtful comments and hope that the\nrevised manuscript now meets the high standards of Nature Communications.\nReviewer #3 (Remarks to the Author):\nYang et al., report a structure of an ammonia monooxygenase enzyme (AMO complex)\nfrom the ammonia-oxidising bacterium (AOB) Nitrosomonas halophila, and from a\nnew strain that the authors isolated themselves from mangrove sediments. They have\nengineered a tagged version of the subunit Amo-A that they have used to isolate the full\ncomplex that they have analysed by cryo-EM. At the end of 2024, another group also\nobtained the structure of an AMO complex of the same family, Nitrosomonas europaea\n(Tucci et al., 2024). This study by Yang et al. is only slightly incremental compared to\nTucci et al, as they manage to find the identity of the long transmembrane helix AMOD\nand to visualise a new AMOE short helical protein. The cryo-EM maps look nice, but\nthe manuscript falls short of explanations and insights all through the text.\nReply: We thank the reviewer for their thoughtful assessment of our work. We are\npleased to highlight that our study provides several key advances beyond the structure\nreported by Tucci et al. (2025), offering substantial new insights into the architecture\nand mechanism of ammonia monooxygenase (AMO).\nFirst, our structural analysis revises the oligomeric organization of AMO, revealing that\nit assembles as a homotrimer of heteropentamers (each composed of AmoA, AmoB,\nAmoC, AmoD and AmoE), rather than a heterotetramer. This refined architectural\nmodel may have significant implications for understanding the complex’s stability,\ncatalytic function, and regulatory mechanisms, particularly in light of the proposed\nfunctional roles of AmoD and AmoE inferred from their genomic contexts.\nSecond, we present the first direct structural evidence for the simultaneously occupied\nCu and Cu sites. Recent quantum mechanics/molecular mechanics (QM/MM)\nC D\nsimulations have demonstrated that both Cu and Cu sites tend to maintain the Cu (I)\nC D\nactive state, highlighting the potential relevance of a dicopper state; however, this state\nhad not been structurally resolved6. This finding of a dicopper center provides a\nfundamental structural basis for understanding the diversity of the AMO active site and\nthe complexity of its catalytic mechanism.\nThird, based on our high-resolution map, we propose a plausible proton transfer\npathway from Cu to Cu site within AMO, offering mechanistic hypotheses that can\nB D\nbe tested in future functional studies.\nFourth, we identify pronounced differences in surface electrostatic potential between\nAMO and pMMO, suggesting a structural basis for their distinct substrate selectivity\n(ammonia vs. methane).\nTo enhance the physiological relevance of our structural findings, we have undertaken\nseveral supplementary experimental investigations (please refer to the following text\nfor details):\n1) We re-purified the AMO sample, collected additional cryo-EM data, and determined\na new AMO structure with the resolution of 2.59 Å, thereby confirming the presence of\nwater molecules and lipids, and enhancing the accuracy of the structural model;\n2) We have identified the potential electron donor of AMO and may facilitate the\ndetection of enzymatic activity in future investigations.\n3) We conducted X-ray photoelectron spectroscopy (XPS) to detect copper within the\nAMO complex and to rule out the potential presence of zinc;\n4) We carried out site-directed mutagenesis on key residues involved in copper\ncoordination, followed by enzyme activity assays to assess their functional roles;\n5) We conducted lipidomics analysis on both the purified AMO sample and the newly\nisolated AOB strain to refine the lipid modeling in the structural study.\nWe have fully revised the manuscript to improve clarity, emphasize mechanistic\ninsights, and better highlight these conceptual and experimental advances. We believe\nour work provides a deeper and more accurate structural and functional understanding\nof AMO that significantly extends beyond the current literature.\nThe introduction could be better and more readable by explicitly explaining some\nconcepts the are mentioned, but are not known by readers that are not in the field of\nmono oxygenates. As an example in line 6 the authors write “consisting\n6 of three components and controversial copper-binding sites”, they could explain what\nthis controversy is about. Does it have to do with the fact that in some of the pMMO\nstructures Zn instead of Cu is found in the metal centers?\nReply: We thank the reviewer for this helpful suggestion. We have thoroughly revised\nthe introduction to improve its clarity and accessibility, particularly for readers outside\nthe field of monooxygenase enzymology.\nSpecifically, regarding the controversy over copper-binding sites mentioned in the\nmanuscript, we have expanded the explanation to provide necessary context. The debate\nprimarily stems from discrepancies in the identity, location, and nuclearity of the\ncatalytic copper site in homologous particulate methane monooxygenase (pMMO). For\ninstance, some early pMMO crystal structures reported zinc in metal-binding\npositions—likely an artifact introduced by the use of ZnSO during crystallization. This\n4\nhas led to ongoing discussions concerning the physiologically relevant metal centers in\nboth pMMO and AMO.\nWe have added a concise summary of this controversy in the introduction as follows:\n“Over two decades of structural investigations have unveiled the α3β3γ3 trimeric\narrangement of pMMO, which consists of three protein subunits: PmoA, PmoB, and\nPmoC. While several copper ligand environments have been identified in pMMO, the\nidentity of the true active site has long been a subject of debate. The bis-His site (Cu )\nA\nhas historically been considered unlikely to be the active center due to the lack of\nconservation in coordinating residues. Previous extended X-ray absorption fine\nstructure (EXAFS) data indicated the dinuclear feature of Cu-Cu scattering interaction\nin Cu site, subsequently high-resolution structures showed a mononuclear Cu site.\nB B\nGiven that Cu presents as Cu(II) and has no obvious hydrophobic pocket for substrate\nB\nbinding. Further, the mutation of one of the Cu ligands in a related hydrocarbon\nB\nmonooxygenase did not completely abolish activity. Consequently, scientists currently\ntend to believe that the Cu site is not an active site. The earlier crystal structures\nB\nrevealed that zinc ions occupy the Cu site due to the artificial addition of crystallization\nC\nreagents ZnSO . However, its strict conservation and completely abrogated activity\n4\nupon mutation of coordinated residues suggest that the Cu\nC\nis the likely active site.\nRecently, the presence of the Cu site, the binding of a substrate analog near the Cu\nD D\nand the electron nuclear double resonance data supported the notion that the Cu is the\nD\nactive site. However, quantum mechanics/molecular mechanics (QM/MM) simulations\nsuggest that both Cu and Cu tend to remain in the Cu(I) active state6, highlighting the\nC D\npotential relevance of a dicopper state. Given that the short distance between Cu and\nC\nCu (~5.7 Å resolved in previous pMMO structures containing single Cu or Cu ), the\nD C D\npossibility of co-occupied Cu and Cu sites remains open8 ”.\nC D\nThese revisions are intended to provide a clearer and more balanced background,\nfacilitating a broader understanding of the structural and functional questions addressed\nin our study.\nRegarding this, we have a major concern, how do the authors know that they have Cu\nrather than Zn bound in their structure? By EM density analysis they cannot be certain\nabout it. We suggest to perform some sort of X-ray spectroscopy to identify the element.\nThe authors mention in line 14 that AMO has been established to be a Cu dependent\noxygenase, but there is no reference. This lack of citations is spread all over the text. It\nis hard to follow for someone that doesn’t know the specific literature.\nReply: We thank the reviewer for raising this critical point regarding metal identity and\nthe need for supporting references throughout the manuscript.\nIn direct response to the reviewer’s suggestion, we performed X-ray photoelectron\nspectroscopy (XPS) analysis on our purified AMO sample. The results show a weak but\ndetectable copper signal, suggesting the presence of copper in AMO sample. No zinc\nsignal was detected throughout the measurement, indicating the absence of zinc in\nAMO sample (Response Figure 3). The low intensity of the copper signal maybe\nattributed to the following factors:\n(1) Release of copper from AMO: To rule out interference from the copper ions in the\nbuffer as much as possible, we used the copper-free buffer to purify the AMO sample,\ngiven the potentially low binding affinity of copper and AMO (binding energy > 930\neV), and the dynamic equilibration of copper between AMO and buffer, the copper\nligands are highly likely to release from the AMO during protein purification.\n(2) Location of the copper ligands: The copper ions in our protein are deeply embedded\nwithin the large, multi-subunit transmembrane structure. The sampling depth of XPS is\ntypically limited to 5-10 nm. Therefore, the photoelectrons (Cu 2p) generated from\nthese internal copper atoms are highly likely to be inelastically scattered and absorbed\nby the surrounding protein matrix and lipid bilayer before they can escape the surface\nand be detected.\n(3) Low abundance of copper in AMO: The stoichiometry of copper in AMO complex\nis relative low (only 3 coppers per AMO protomer). Consequently, the photoelectron\nsignal originating from copper is intrinsically weak compared to the abundant carbon,\nnitrogen, and oxygen signals from the protein itself.\nAlthough the current experimental technique and sample require further optimization,\nour results provide direct evidence for the identification of copper in AMO.\nResponse Figure 3. XPS analysis of the metal composition in AMO. The core-level\nspectra of Cu 2p (a) and Zn 2p (b) are displayed, with characteristic peaks labeled as\nCu 2p and Cu 2p , corresponding to distinct electronic states of copper. No\n1/2 3/2\ndetectable signal from zinc was observed.\nIn addition, we also acknowledge and apologize for the previously insufficient citation\nof relevant literature. We have now thoroughly reviewed the entire text and added\nappropriate references throughout the revised manuscript to better support key\nstatements—including the copper-dependent nature of AMO—and to improve clarity\nfor readers outside the field.\nWe believe these revisions have significantly strengthened the manuscript and thank\nthe reviewer for their constructive suggestion.\nThe main text, results and discussion, is a description of the structure, but lacks\ncompletely functional analysis and argumentation about the role of certain observations.\nWe consider and understand that in vitro biochemical characterisation of such en\nenzyme is very difficult, but some level of in-cell functional study should be possible\nand would make the paper more valuable. The authors could manipulate the bacteria\nstrain to make a AMO-A tagged version. Is it not possible to include mutations in the\nvarious metal centres and then check the level N2O formation?\nThis would be really necessary to prove that the water path is important for e- transfer\n(as the authors state) and could be used to prove the physiological relevance of the\ndouble-copper presence at the Cu-D and Cu-C site.\nWithout any other type of assay, the structure analysis remains quite sterile,\nunfortunately.\nReply: We thank the reviewer for emphasizing the importance of functional studies to\ncomplement our structural findings. We fully agree and have conducted additional\nmutagenesis experiments to directly address this point.\nA significant advance in our study—previously underemphasized—is the development\nof a genetic manipulation system for Nitrosomonas halophila ZJB1. Although the\nsystem currently has limitations (e.g., low transformation efficiency and slow screening\ndue to low growth rates of AOB), it provides a critical tool for functional dissection of\nAMO.\nUsing this system, we introduced mutations into residues coordinating the Cu and Cu\nC D\nsites. We successfully generated strains with single-copy mutations in H140A,\nH153A, D146A, N207A, E218A, and H211A. Attempts to construct D136A and H225A\nmutants were unsuccessful, likely because these mutations severely impair function and\nare lethal. Following the reviewer’s suggestion, we measured ammonia consumption—a\ndirect readout of AMO activity—in these mutants. Activity assays revealed a\nmeasurable reduction in ammonia oxidation activity in these mutants, with decreasing\nactivity by approximately 30% (Response Figure 2a). The moderate reduction is likely\ndue to the presence of three genomic copies of the amo operon, which may partially\ncompensate for the loss of function in a single copy. These functional effects are\nconsistent with observations from the previous study of pMMO subunit deletions5\n(Response Figure 2b-c), supporting the essential role of these residues in catalytic\nactivity. This portion of the results has been integrated into the revised Figure 2g.\nResponse Figure 2. Determination of the enzymatic activity rates in AMO single-\nmutant strains. This figure illustrates the alterations in enzymatic activity resulting\nfrom mutations in key residues involved in coordinating Cu and Cu within AMO, as\nC D\nobserved in this study (a), in comparison to previously reported data for deletions of\nAmoA1 (■) and AmoA2 (▲) (b), and AmoB1 (■) and AmoB2 (▲) (c). Wild-type\nactivity is indicated by (●)5.\nThe reviewer also rightly highlighted the need to test the functional role of the proposed\nwater-mediated hydrogen-bond network. We attempted to mutate key residues (e.g.,\nW139A, I143A) involved in this network. Despite multiple attempts, we have not yet\nobtained these mutants, suggesting that these residues may be essential for bacterial\nviability or ammonia oxidation process. We are continuing to optimize genetic\nconditions to overcome this, though obtaining such mutants may require further time\nand method development.\nWe have included the new mutational data in the revised manuscript and have softened\nour description of the water network, clearly stating that it represents a proposed\nmechanistic model requiring further functional validation.\nWe are grateful for these constructive comments, which have greatly enhanced the\nfunctional depth of our study.\nThe analysis of the lipids and their distribution is completely relegated to the\nsupplementary materials, not attempt to identify the type of lipids has been done (e.g.\nmass spec?) and the lipids are not built in the deposited pdb. Why? We noticed that\nalso the other group’s structure (Tucci et al. pdb-9cl6) doesn’t contain the lipids. On\nthe other hand, very many water molecules were built in the maps, which is a bit\nsurprising at this resolution. For some water molecules there is no density and for other\nwater molecules the density is ambiguous and might also represent noise. The\nhigh-resolution noise and intrinsic motion of the outer side of the cryo-EM maps,\nmakes it difficult to believe that all those H2O molecule can be built in the periphery of\nthe structure. In the supplementary part about the methods the author are completely\nmissing the description about model building. This should be corrected. We wonder\nwhether the model was build starting from previous homologous structures that already\nincluded the waters. In any case the procedure that was followed to build the model\nshould be described.\nReply: We thank the reviewer for these important comments, which have helped us\nimprove the clarity and rigor of our structural model. Below we provide a point-by-\npoint response.\n1. Regarding the modeling of lipids:\nWe acknowledge that lipids were not included in the initial PDB deposition due to the\nrelatively weak and flexible density of lipid acyl chains, which made unambiguous\nmodeling difficult at the current resolution—consistent with the approach taken in the\npreviously reported AMO structure (Tucci et al. PDB: 9CL6). In direct response to the\nreviewer’s suggestion, we have now performed lipidomics mass spectrometry to\nidentify the lipid composition of our AMO sample. Results showed that the most\nabundant lipid species were free fatty acid (FFA), monoglyceride (MG), diglyceride\n(DG), and phosphatidylserine (PS) (Response Figure 7). This portion of the results has\nbeen incorporated into the revised Fig. 1d and Supplementary Fig. 6. Based on this\nspectrometric evidence, we have modeled 84 lipid molecules into the structure,\nincluding 60 FFA, 6 MG, 6 PS, 3 DG and 9 phosphatidylmethanol (PMeOH) (Response\nFigure 8). The updated structural model, including these lipids, will be deposited in the\nPDB and described in the main text, with corresponding visual representation provided\nin the revised Fig. 1e.\nResponse Figure 7. Analysis of lipidomics data of purified AMO protein (a) and\nNitrosomonas halophila ZJB1 cell (b). Lipids are sorted by head group type, degrees\nof unsaturation in lipid tails, and lipid tail lengths. Types shown are free fatty acid (FFA),\nmonoglyceride (MG), diglyceride (DG), phosphatidylserine (PS), phosphatidyl\nethanolamine (PE) and other lipids with lower abundance.\nResponse Figure 8. Representative lipids modeled in AMO structure. The\ncorresponding electron density maps are displayed in transparency.\n2. Regarding the modeling of water molecules:\nTo validate the water molecules placed in the model and to rule out the possibility\ncaused by noise, we performed an independent replicate experiment. We repurified the\nAMO complex, collected a new cryo-EM dataset, and determined a new structure at\n2.59 Å resolution which is comparable to the original 2.36 Å. The density features\ncorresponding to water molecules were consistently observed (Response Figure 9). This\nresult strongly confirms that these densities are not artifacts of noise. Additionally, the\nplacement of these water molecules is also supported by previously reported high-\nresolution structures of pMMO (e.g., PDB: 7S4H) and the recently published AMO\nstructure (PDB: 9CL6). We are therefore confident that these solvent molecules are well\njustified and not the result of noise. To further enhance the reliability of the model, we\nretained only those water molecules that are consistently observed in both structures\nand removed certain peripheral water molecules whose densities were relatively weak\nor poorly defined in either structure.\nResponse Figure 9. Validation of water molecules in the AMO structure. a\nSchematic workflow for cryo-EM data processing of the newly prepared AMO sample.\nb Fourier Shell Correlation (FSC) curve of the newly determined AMO structure,\nindicating an overall resolution of 2.59 Å based on the FSC = 0.143 criterion. c\nComparative analysis of water molecules distribution between the original AMO\nstructure (resolution 2.36 Å, shown in gray) and the newly refined AMO structure\n(resolution 2.59 Å, shown in yellow). The majority of water molecules are localized at\nhighly overlapping positions in both structures, confirming that the observed water\nmolecule density is not attributable to noise.\n3. Regarding model building:\nWe apologize for the omission of detailed model-building methods in the original\nsubmission. The atomic model was built de novo using the cryo-EM map, the protein\nsequence, and predictions from AlphaFold. The modeling process did not rely on\npreviously solved homologous structures, as no such structures were available at the\ntime. We have now included a comprehensive description of the model building,\nrefinement, and validation process in the Methods section as follows:\n“Model building and refinement\nThe predicted AlphaFold structures of AmoA, AmoB, AmoC, AmoD, and AmoE were\nmanually docked into the cryo-EM density map using UCSF Chimera and subsequently\nadjusted in Coot according to the corresponding density and sequence information.\nLipid molecules were selected based on lipidomics data and identifiable features, and\nwere manually fitted into the map using Coot. Water molecules within the AMO\ncomplex, particularly those near copper centers, were modeled with reference to the\npreviously reported pMMO structure (PDB: 7S4H) and supported by mass\nspectrometry data. Additional cytoplasmic and periplasmic water molecules were\nplaced based on the pMMO structure (PDB: 7S4H) and validated by the recently\npublished AMO structure (PDB: 9CL6). Real-space refinement and validation of the\nfinal model were carried out using Phenix. Structural figures were prepared with UCSF\nChimera, ChimeraX, and PyMOL (http://www.pymol.org).”\nWe hope these revisions address your concerns and improve the rigor and transparency\nof our structural analysis. Thank you again for your valuable feedback.\nGoing through the text:\n- Page 3 Line 13: which are the two genes downstream of amoCAB that were\nconsidered accessory components of AMO in AOB?\nReply: We thank the reviewer for pointing out this lack of clarity. The two genes located\ndownstream of amoCAB, which have been proposed to serve as accessory components\nof the AMO complex in some ammonia-oxidizing bacteria, are orf1 and orf5 9.\nWe have revised the manuscript to explicitly name these genes and include the\nappropriate citation at the indicated location.\n- Page 4, line 6: what do the authors mean by “components”?\nReply: We thank the reviewer for requesting this clarification. The term “components”\nrefers to the three protein subunits—PmoA, PmoB, and PmoC—that together form a\nprotomer of the particulate methane monooxygenase (pMMO) complex. We have\nrevised the sentence in the manuscript to read:\n“Over two decades of structural investigations have unveiled the α3β3γ3 trimeric\narrangement of pMMO, consisting of three components—PmoA, PmoB and PmoC.”\nThis revision aims to improve clarity and avoid ambiguity.\n- As said before, mutagenesis of the key residues identified would be really useful to\ngain mechanistic information\nReply: We thank the reviewer for emphasizing the importance of mutagenesis studies\nfor mechanistic insight. In response, we have performed site-directed mutagenesis on\nkey residues involved in copper coordination (Cu , and Cu ), including H140A,\nC D\nH153A, D146A, N207A, E218A and H211A.\nActivity assays revealed a measurable reduction in ammonia oxidation activity in these\nmutants, with activity decreasing by approximately 30% (Response Figure 2). The\nmoderate reduction is likely due to the presence of three genomic copies of the amo\noperon, which may partially compensate for the loss of function in a single copy. These\nfunctional effects are consistent with observations from the previous study of pMMO\nsubunit deletions5 (Response Figure 2b-c), supporting the essential role of these\nresidues in catalytic activity.\nThis portion of the results has been integrated into the revised Figure 2g.\nResponse Figure 2. Determination of the enzymatic activity rates in AMO single-\nmutant strains. This figure illustrates the alterations in enzymatic activity resulting\nfrom mutations in key residues involved in coordinating Cu and Cu within AMO, as\nC D\nobserved in this study (a), in comparison to previously reported data for deletions of\nAmoA1 (■) and AmoA2 (▲) (b), and AmoB1 (■) and AmoB2 (▲) (c). Wild-type\nactivity is indicated by (●)5.\nThese results provide direct experimental evidence that the identified copper-\ncoordinating residues are critical for AMO function, offering valuable mechanistic\ninsights into the enzyme’s catalytic mechanism.\n- Page 8 Line 8an almost 4Å bond is labelled as H-bond this is a bit too far away\nReply: We thank the reviewer for this accurate observation. A distance of nearly 4 Å\nexceeds the conventional range for a typical hydrogen bond. We have therefore revised\nthe description from “H-bond” to “weak interaction” in the manuscript to reflect this\nmore appropriately.\n- Page 8 Line 16-17 The authors compare one TM helix in their complex with the\ncorrespondent one in pMMO. It would be useful to have a sequence alignment to see\nhow conserved this helix is and if there are specific aa that would explain the kink of\nthe helix.\nReply: We thank the reviewer for this excellent suggestion. In response, we performed\na detailed sequence alignment of the TM5 helix in AMO and pMMO. The alignment\nreveals a high degree of conservation, particularly in key residues involved in Cu\nD\ncoordination and those situated at the kink region of the helix (Response Figure 10).\nThis conservation suggests a functionally important role for TM5 in both AMO and\npMMO.\nThis portion of the results has been integrated into the revised Figure 2h-i.\nResponse Figure 10. Conservation analysis of TM5. a The Cu and Cu sites are\nC D\nlocated in the upper portion of the hydrophobic channel formed by five helices.\nCompared to pMMO and AMO variants containing a single copper ligand, the outward\ndisplacement of TM5 in the AMO structure with simultaneously occupied Cu and Cu\nC D\nligands determined in this study leads to a substantial increase in the volume of the\nhydrophobic channel. b Sequence alignment of TM5 from representative ammonia- and\nmethane-oxidizing species reveals highly conserved residues at the Cu site and the\nD\nkink region of TM5.\nWe have revised the manuscript to incorporate this analysis and updated the description\nas follows:\n“Sequence alignment demonstrates that TM5 is conserved among AOB and methane-\noxidizing bacteria (MOB). Notably, the histidine and asparagine residues coordinating\nCu , as well as the residues at the kink site, are highly conserved (Fig. 2i), indicating a\nD\nshared role in regulating the Cu site and hydrophobic channel in both AMO and\nD\npMMO.”\n- Line 8-12 page 9 the manuscript reads:\nClose inspection of three coppers’ coordination environment reveals that a relatively\nweak hydrogen bond (Fig. 2b, e and f, red stars) consistently accompanies these coppers,\nsuggesting their potential role in facilitating electron transfer during substrate oxidation,\nas previously reported, beyond merely stabilizing the copper site44-46.\nHow does the hydrogen bond network facilitate electron transfer? Could you please\ngive a reference for such mechanism? Could the electrons not tunnel directly from metal\ncenter to metal center given that the distance is less than 20 Å? Do the authors maybe\ninstead mean proton transfer to the active site?\nReply: We sincerely thank the reviewer for this critical and insightful comment. The\nreviewer is absolutely correct. Upon re-evaluation, we agree that the role of the\nhydrogen-bonding network is much more consistent with facilitating proton transfer\nrather than electron transfer. Electrons can indeed tunnel directly between metal centers\nover these distances, while protons require such a structured network for efficient\ntransfer via the Grotthuss mechanism10.\nWe have revised the manuscript accordingly to correct this important concept. The\nrelevant text now clearly proposes a role for the observed hydrogen bonds in proton\ntransfer, and we have provided appropriate references to support this mechanism11-13.\nWe appreciate the reviewer’s guidance, which has significantly improved the accuracy\nof our discussion.\n- Page 9, line 15 the rod-shaped density could also represent noise instead of\nhydroxylamine\nReply: We thank the reviewer for raising this valid concern regarding the rod-shaped\ndensity. We agree that at the current resolution, its identity cannot be unambiguously\ndetermined. To evaluate whether this feature represents noise, we performed an\nindependent replicate experiment: we repurified the AMO complex, collected a new\ncryo-EM dataset, and determined a new structure at 2.59 Å resolution which is\ncomparable to the original 2.36 Å. The rod-shaped density was consistently observed\nin the same location (Response Figure 11), suggesting it is a reproducible structural\nfeature rather than random noise.\nResponse Figure 11. Validation of the rod-shaped density. The rod-shaped densities\nin the original map (a) and the newly determined map (b) are presented.\nBased on its location within the proposed active site and the known ammonia oxidation\npathway, we hypothesize that this density may correspond to a substrate or reaction\nintermediate. However, we fully acknowledge that further experimental evidence is\nrequired for definitive identification.\nIn response to the reviewer’s comment, we have revised the relevant description in the\nmanuscript to more accurately reflect this interpretation:\n“Interestingly, an elongated, rod-shaped density is observed in the hydrophobic channel\nadjacent to the Cu –Cu center (Fig. 2c). While its identity remains uncertain, its\nC D\nlocation suggests a potential role in substrate access or product release. Further studies\nat higher resolution or under different catalytic conditions are necessary to elucidate the\nnature of this feature.”\n- Page 10 Line 1 it is confusing that the bis-His site in introduced all of a sudden with\nno context, nor it was given in the introduction and with references lacking.\nReply: We thank the reviewer for this important observation. In response, we have\nexpanded the Introduction to provide a clearer background on the historical and current\nunderstanding of copper sites in pMMO, including the bis-His site and the ongoing\ndebate surrounding the identification of the genuine active site. Relevant references\nhave been added to support this discussion. The revised text now reads:\n“While several copper ligand environments have been identified in pMMO, the identity\nof the authentic active site has long been a subject of debate. The bis-His site (Cu ) has\nA\nhistorically been considered unlikely to be the active center due to the lack of\nconservation in coordinating residues. Previous extended X-ray absorption fine\nstructure (EXAFS) data indicated the dinuclear feature of Cu-Cu scattering interaction\nin Cu site, subsequently high-resolution structures showed a mononuclear Cu site.\nB B\nCu presents as Cu(II) and has no obvious hydrophobic pocket for substrate binding.\nB\nFurther, the mutation of one of the Cu ligands in a related hydrocarbon\nB\nmonooxygenase did not completely abolish activity. Consequently, scientists currently\ntend to believe that the Cu site is not an active site. The earlier crystal structures\nB\nrevealed that zinc ions occupy the Cu site due to the artificial addition of crystallization\nC\nreagents ZnSO . However, its strict conservation and completely abrogated activity\n4\nupon mutation of coordinated residues suggest that the Cu is the likely active site.\nC\nRecently, the presence of Cu site, the binding of substrate analogue near the Cu and\nD D\nthe electron nuclear double resonance (ENDOR) data supported the Cu being the\nD\nactive site. However, quantum mechanics/molecular mechanics (QM/MM) simulations\nsuggest that both Cu and Cu tend to remain in the Cu(I) active state, highlighting the\nC D\npotential relevance of a dicopper state. Given that the short distance between them (~5.7\nÅ resolved in previous pMMO structures containing single Cu or Cu ), the possibility\nC D\nof co-occupied Cu and Cu sites remains open8.”\nC D\n- Page 10 Line 14-16 . Notably, AmoD is positioned adjacent to AmoC with its N-\nterminal facing the cytoplasmic side and C-terminal oriented towards the periplasmic\nside (Fig. 1 and 3a).\nWhy notably? What is the functional implication of this observation?\nReply: We thank the reviewer for raising this valid point. The word “notably” has been\nremoved in the revised manuscript.\nAnd again on then following page The first alkyl chain of this lipid interacts with AmoD\nvia Trp37 and penetrates into the hydrophobic core of AmoD and AmoC, while the\nsecond alkyl chain of this lipid anchors onto the surface of the TM1 of AmoC and\nstretches into the lipid bilayer of the cell membrane (Fig. 3c).\nWhat are the physiological implications of such observation?\nReply: We thank the reviewer for this insightful question regarding the functional\nrelevance of the lipid binding motif. The specific interaction, in which one alkyl chain\npenetrates the hydrophobic core between AmoD and AmoC while the other extends into\nthe lipid bilayer, suggests a dual structural and functional role. We have expanded the\ndiscussion in the revised manuscript as follows:\n“The first alkyl chain of DG interacts with AmoD through Trp37 and penetrates the\nhydrophobic core formed by AmoD and AmoC. Concurrently, the second alkyl chain\nanchors onto the surface of TM1 in AmoC and extends into the lipid bilayer of the cell\nmembrane. This distinct lipid-binding mode suggests that these interactions may\ncontribute to stabilizing the structural integrity of the AMO complex and facilitating\nthe proper integration and localization of AmoD within the membrane environment.”\n- Based on the bioinformatics analysis a possible role is assigned to AMO-D, but it is\nunclear. How is ammonia oxidation related to the TCA cycle? This is most likely due\nto our ignorance, but it would be better of the authors would explain the correlation\nbetween the two enzymatic processes.\nReply: We thank the reviewer for raising this insightful question regarding the potential\nfunctional link between AmoD and the TCA cycle, which allows us to clarify a key\npoint in our mechanistic hypothesis.\nOur initial genomic analysis indicated that the gene encoding AmoD is located near\ngenes involved in the TCA cycle and ATPase function. This bioinformatic observation\nled us to speculate a possible functional connection. Crucially, our subsequent\nbiochemical experiments identified CoQ H as a potentially effective electron donor\n10 2\nfor AMO, which may facilitate the detection of enzymatic activity in future\ninvestigations.\nThis discovery provides a mechanistic link between AmoD and central metabolism.\nThe TCA cycle generates reducing equivalents (NADH and FADH ), which are used to\n2\nreduce membrane-bound CoQ to ubiquinol (CoQ H ). We propose that this ubiquinol\n10 10 2\npool then serves as the direct electron donor for AMO. Therefore, the activity of AMO\nis indirectly coupled to the TCA cycle through the shared use of this common redox\ncarrier (CoQ /CoQ H ). The genomic association of amoD with TCA cycle genes may\n10 10 2\nreflect a role for AmoD in regulating or facilitating this electron supply from central\nmetabolism to the AMO complex, ensuring optimal enzyme activity.\nWe have revised the manuscript to clarify this possible physiological relationship as\nfollows:\n“We propose that AmoD may play a role in coordinating electron flow from central\nmetabolism to AMO. This model is supported by our identification of CoQ H —a\n10 2\nmetabolic electron carrier regenerated by TCA cycle-derived reducing equivalents—as\nan effective electron donor for AMO (Supplementary Fig. 3c-d). Therefore, AmoD may\nfacilitate metabolic coupling to maintain AO activity under diverse physiological\nconditions.”\nThis interpretation remains hypothetical, but it offers a testable model for future studies\non the bioenergetic integration of ammonia oxidation within bacterial metabolism.\n- It is unclear why the positive electrostatic charge distribution at the membrane levels\nshould play a very important role.\nReply: We thank the reviewer for raising this important point. We agree that the\npresence of positively charged regions facilitating the transport of a positively charged\nion such as NH ⁺ appears counterintuitive at first glance. Below we clarify our\n4\nreasoning, which draws on electrostatic considerations, the biochemical properties of\nammonium, and a mechanistic precedent:\nElectrostatic steering via alternating charge patterning:\nThe positive charges should not be considered in isolation; rather, they form part of an\nalternating electrostatic motif along the membrane interface. In this model: Negatively\ncharged residues attract NH ⁺ ions from the extracellular milieu. Adjacent positive\n4\nresidues help lower the energy barrier for ion permeation by electrostatically shielding\nthe NH ⁺ ion from subsequent repulsive negative patches, thereby facilitating a stepwise\n4\n“hand-off”. Thus, the positive charges serve not as direct attractors, but as facilitators\nthat mitigate electrostatic repulsion and reduce the dielectric barrier for ion\ntranslocation.\nPossible deprotonation of NH ⁺ to NH :\n4 3\nThe ammonium–ammonia equilibrium (NH ⁺ ⇌ NH + H⁺) offers another plausible\n4 3\nmechanism. The local environment near the membrane—potentially modulated by\nnegative charges—could promote deprotonation, forming neutral NH . This uncharged\n3\nspecies would encounter no Coulombic barrier from positive patches and may diffuse\nreadily through hydrophobic regions. Reprotonation could subsequently occur near the\nactive site. In this scenario, positive surface charges help form a permissive path for\nNH .\nThis electrostatic guidance mechanism may be analogous to that recently observed in\ncation-translocating membrane proteins, the eukaryotic magnesium channel Mrs214,\nwhere a charged vestibule facilitates the selective recruitment of cations from the\nenvironment (Response Figure 12). This supports the idea that such an arrangement is\na functional and evolutionarily relevant feature in cation-translocating membrane\nproteins.\nIn summary, we propose that the positive charges help form a dynamic electrostatic\nnetwork that guides NH ⁺ ingress—either via charge shielding or by permitting NH\n4 3\ndiffusion. We acknowledge that further functional studies are needed to fully establish\nthe biological role of this electrostatic feature and have toned down speculative\nconclusions accordingly.\nResponse Figure 12. Potential function of the positive/negative electrostatic charge\ndistribution at the membrane interface of membrane-bound proteins transporting\npositively charged substrates. The surface electrostatic potentials of AMO (a) and\nMrs2 (b) were shown.\n- The conclusions seem a bit misleading and not completely fair. First the assembly is\ndescribed as unexpected while it cannot be the case view that the other AMO structure\nfrom Tucci et al, was already available and then they conclude with a comment about\nthe negative aspects of the structure from the other lab instead of focusing on what\ncould have been novel in their presented structure.\nReply: We thank the reviewer for this constructive feedback regarding the framing of\nour conclusions.\nWe acknowledge that the term “unexpected” was inappropriate, particularly in light of\nthe concurrent work by Tucci et al. This wording has been removed from the revised\nmanuscript. We have also reframed our discussion to focus on the novel aspects of our\nstructure—such as the identification of the simultaneously occupied Cu –Cu site, the\nC D\nincorporation of AmoE, and the distinct electrostatic features—rather than comparing\nperceived limitations of the other study.\nWe believe these changes provide a clearer and more collegial perspective on the\nadvancing structural understanding of AMO.\nTaking all this into account, we suggest that a substantial effort is put by the authors in\nsome assays that would highlight the physiological importance of their structural\nobservations. Moreover, the text is missing crucial references in several places (for\nexample Page 7, line 16, a reference to the comparison AMO structure is needed). Such\nreferences should be put all throughout the text.\nReply: We sincerely thank the reviewer for these critical and constructive comments,\nwhich have significantly helped us improve the depth and rigor of our manuscript.\nIn direct response to the suggestion to strengthen the physiological relevance of our\nstructural findings, we have performed the following additional experiments:\n1) We determined a new cryo-EM structure of the AMO complex at 2.59 Å resolution\nfrom an independently purified sample. This replicate structure confirms the presence\nof key water molecules and lipids, enhancing the reliability of our model.\n2) XPS analysis suggested the presence of copper and ruled out zinc incorporation at\nthe metal centers of AMO.\n3) We identified the potential electron donor of AMO and may aid in the detection of\nenzymatic activity in future studies;\n4) We performed site-directed mutagenesis on residues coordinating the copper sites\nand measured enzymatic activity, revealing a decrease in enzyme activity that supports\ntheir essential roles.\n5) Lipidomics analysis of the native membrane and purified AMO complex enabled\naccurate identification and modeling of associated lipids.\nFurthermore, we have thoroughly revised the manuscript to include crucial references\nthroughout, particularly adding a citation for the comparative AMO structure (Tucci\net al.) at the appropriate locations.\nWe believe these extensive revisions have substantially strengthened the physiological\nand mechanistic insights of our study and greatly improved the scholarly context of the\nwork.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReply: Thank you for your participation in the review of our manuscript and for\ncontributing to the peer-review process as part of Nature Communications’ initiative to\nsupport early career researchers. We greatly appreciate the time and effort you have\ndedicated to evaluating our work.\nYour feedback, along with that of your colleague, has been carefully considered and\nhas helped us improve the quality and clarity of our manuscript. We have made\nrevisions accordingly and believe the study has been strengthened as a result.\nWe are grateful for your involvement in this process.\nReferences\narchaeal surface layer proteins. Isme J 12, 2389-2402 (2018).\nhttps://doi.org/10.1038/s41396-018-0191-0\nin archaea. The ISME Journal 17, 588-599 (2023).\nhttps://doi.org/10.1038/s41396-023-01367-3\nprophenoloxidase (PPO) from provides new insights into the mechanism of\nPPO activation. Bmc Biol 14 (2016). https://doi.org/ARTN 210.1186/s12915-\n015-0225-2\n3659-3853 (2014). https://doi.org/10.1021/cr400327t\nregulation of amoA and amoB gene copies in Nitrosomonas europaea. Fems\nMicrobiol Lett 192, 163-168 (2000). https://doi.org/DOI 10.1111/j.1574-\n6968.2000.tb09376.x\nValence State and Reactivity of Copper Centers in Membrane-Bound\nParticulate Methane Monooxygenase. J Am Chem Soc 145, 25304-25317\n(2023). https://doi.org/10.1021/jacs.3c08834\nmonooxygenases in native membranes. P Natl Acad Sci USA 122 (2025).\nhttps://doi.org/ARTN e241799312110.1073/pnas.2417993121\nIron-Dependent Methane Monooxygenases. Chemical Reviews 124, 1288-1320\n(2024). https://doi.org/10.1021/acs.chemrev.3c00727\nnew genes, amoR and amoD, in the operon of the marine ammonia oxidizer\nNitrosococcus oceani ATCC 19707. Applied and Environmental Microbiology\n74, 312-318 (2008). https://doi.org/10.1128/Aem.01654-07\nwires. Isr J Chem 39, 387-395 (1999).\nMolecular Principles of Redox-Coupled Protonation Dynamics in Photosystem\nII. J Am Chem Soc 144, 7171-7180 (2022).\nhttps://doi.org/10.1021/jacs.1c13041\nevolution. Nature 617 (2023). https://doi.org/10.1038/s41586-023-06008-5\nwater networks in photosystem II. Science 384, 1349-1355 (2024).\nhttps://doi.org/10.1126/science.adn6541\nMrs2 reveal the auto-ligand-gating regulation mechanism. Nature Structural &\nMolecular Biology 32 (2025). https://doi.org/10.1038/s41594-024-01432-1"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Revising their report on a high-resolution cryo-EM structure of AMO, the authors have taken the reviewers' reports very seriously and in my opinion addressed all points very well and in detail.\nThis is true for my own review, where I have no further points to make and support publication of the manuscript.\nIt is also true, I think, for the other two reviews that both considered the advances made in this work to be only incremental. The authors have responded by providing substantially more functional data with their revision, clearly strengthening the work. From a purely structural perspective, however, and having looked at both the earlier model and the present one reported here, I am convinced that the improvements in electron density quality alone are substantial and very helpful for the field. This already was an excellent piece of structural biology, and it has now improved further.", "reviewer_2": "The authors have addressed the reviewers' previous comments. However, one important clarification remains necessary:\nthe two copper atoms observed in the membrane domain of the Cryo-EM structure (PDB ID: 7EV9) are not located at the\nCuD site, but rather at site D of pMMO, as indicated in Reference 41 and J. Chin. Chem. Soc. 69 (2022) 1147. Furthermore,\nbased on these two sources, the CuC and CuD sites discussed in the present study correspond to site C in pMMO.\nThis reviewer recommends the publication of the manuscript.", "reviewer_3": "The authors have undertaken substational efforts ot improve the manuscript which is now in a form that is much more complete and worth of publication, in our opinion.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the Editor and the Reviewers for their positive evaluation and constructive\ncomments on our manuscript (Research Article, No. NCOMMS-25-30401). We are\ngrateful for the time and effort dedicated to reviewing our work, which has greatly\nhelped us improve the quality and clarity of the paper.\nReviewer #1 (Remarks to the Author):\nRevising their report on a high-resolution cryo-EM structure of AMO, the authors have\ntaken the reviewers' reports very seriously and in my opinion addressed all points very\nwell and in detail.\nThis is true for my own review, where I have no further points to make and support\npublication of the manuscript.\nIt is also true, I think, for the other two reviews that both considered the advances made\nin this work to be only incremental. The authors have responded by providing\nsubstantially more functional data with their revision, clearly strengthening the work.\nFrom a purely structural perspective, however, and having looked at both the earlier\nmodel and the present one reported here, I am convinced that the improvements in\nelectron density quality alone are substantial and very helpful for the field. This already\nwas an excellent piece of structural biology, and it has now improved further.\nReply: We sincerely thank the Reviewer for their positive and supportive feedback on\nour revised manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed the reviewers' previous comments. However, one important\nclarification remains necessary: the two copper atoms observed in the membrane\ndomain of the Cryo-EM structure (PDB ID: 7EV9) are not located at the CuD site, but\nrather at site D of pMMO, as indicated in Reference 41 and J. Chin. Chem. Soc. 69\n(2022) 1147. Furthermore, based on these two sources, the CuC and CuD sites\ndiscussed in the present study correspond to site C in pMMO.\nThis reviewer recommends the publication of the manuscript.\nReply: We thank the Reviewer for their meticulous review and for pointing out the\nmisassignment of the copper sites.\nWe have thoroughly reviewed the relevant literature, including Reference 41 and the\nstudy published in J. Chin. Chem. Soc. 69 (2022) 1147, and have corrected the\nmisassignment of copper ligands observed in the cryo-EM structure (PDB ID: 7EV9),\nas presented in the revised Supplementary Figure 7.\nSupplementary Figure 7 | Summary of copper sites observed in pMMO.\nIn addition, previous cryo-EM structures of pMMO have clearly revealed the presence\nof a single Cu (PDB 7S4J) and a single Cu sites (PDB 7S4H)1 (Response Figure 1b-\nC D\nc), which are distinct from the site D identified in PDB 7EV9 (Response Figure 1a).\nMoreover, a recently published AMO structure also identifies a single Cu site (PDB\nC\n9CL6)2 (Response Figure 1d). Structural alignments demonstrate that the copper\nligands in the dicopper center of our AMO structure correspond to the Cu and Cu\nC D\nsites of pMMO and AMO, respectively (Response Figure 1e-h). Based on these\nstructural comparisons and the conservation of copper-coordinating ligands, we have\nretained the designation of this dicopper center as co-occupied Cu /Cu sites to\nC D\naccurately reflect its composition and structural correspondence to pMMO.\nThank you again for your important clarification, which has significantly improved the\nprecision and rigor of our manuscript. We sincerely appreciate your recommendation\nfor publication.\nResponse Figure 1. Structural comparison of copper-binding sites in pMMO and\nAMO. a-e The copper binding sites in pMMOs structures (a, PDB 7EV9; b, PDB 7S4H;\nc, PDB 7S4J) and AMO structures (d, PDB 9CL6; e, this study). f-h Structural\nalignments of AMO from this study with previously reported pMMO (f, PDB 7S4H;\ng, PDB 7S4J) and AMO (h, PDB 9CL6).\nReviewer #3 (Remarks to the Author):\nThe authors have undertaken substational efforts ot improve the manuscript which is\nnow in a form that is much more complete and worth of publication, in our opinion.\nReply: Thank you for your positive feedback on our revised manuscript. We sincerely\nappreciate your recognition of the substantial efforts we have made to improve the\nmanuscript and your evaluation that the current version is more complete and worthy\nof publication.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReply: Thank you for your participation in the peer review of our manuscript as part of\nthe Nature Communications initiative for Early Career Researchers training. We\nappreciate your time and effort dedicated to the review process.\nReferences\nmethane monooxygenase structure and activity in a lipid bilayer. Science 375,\n1287-+ (2022). https://doi.org/10.1126/science.abm3282\nmonooxygenases in native membranes. P Natl Acad Sci USA 122 (2025).\nhttps://doi.org/ARTN e241799312110.1073/pnas.2417993121"}]

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