Structural basis for late maturation steps of mitochondrial respiratory chain complex IV within the human respirasome

Domain: biology
Published: 2026-01-10
Editorial outcome: minor_revision

Abstract
The mitochondrial respiratory chain comprises four multimeric complexes (CI-CIV) that drive oxidative phosphorylation by transferring electrons to oxygen and generating the proton gradient required for ATP synthesis. These complexes can associate into supercomplexes (SCs), such as the CI+CIII₂+CIV respirasome, but how SCs form, by joining preassembled complexes or by engaging partially assembled intermediates, remains unresolved. Here, we use cryo-electron microscopy to determine high-resolution structures of native human CI+CIII₂+CIV late-assembly intermediates. Together with biochemical analyses, these structures show that respirasome biogenesis concludes with the final maturation of CIV while it is associated with fully assembled CI and CIII₂. We identify HIGD2A as a placeholder factor within isolated and supercomplexed CIV that is replaced by subunit NDUFA4 during the last step of CIV and respirasome assembly. This mechanism suggests that placeholders such as HIGD2A act as molecular timers, preventing premature incorporation of NDUFA4 or its isoforms and ensuring the orderly progression of pre-SC particles into functional respirasomes. Since defects in CIV assembly, including NDUFA4 deficiencies, cause severe encephalomyopathies and neurodegenerative disorders, understanding the molecular architecture and assembly pathways of isolated and supercomplexed CIV offers insight into the pathogenic mechanisms underlying these conditions.

Introduction
The mitochondrial respiratory chain (MRC) consists of four multimeric enzymatic complexes (CI to CIV) and two mobile electron carriers, ubiquinone (Q) and cytochrome c (cytc), which act in concert to drive the transfer of electrons from substrates such as NADH and succinate to molecular oxygen1,2. Electron transfer along the MRC is coupled with the extrusion of protons through CI (NADH:ubiquinone oxidoreductase), CIII (ubiquinol-cytochrome c oxidoreductase), and CIV (cytochrome c oxidase) across the mitochondrial inner membrane. This process establishes a proton gradient that F1o-ATP synthetase (Complex V) uses to synthesize ATP through oxidative phosphorylation (OXPHOS).

Given the central role OXPHOS plays in cellular energy metabolism, deficiencies in the enzymes catalyzing these processes contribute to human diseases. This impact extends to both rare inherited mitochondrial disorders, including Leigh syndrome and cardioencephalomyopathies, and common age-associated diseases3. Therefore, a comprehensive understanding of the assembly, regulation, and functioning of these complexes is crucial for unraveling the mechanisms underlying cellular energy metabolism and its implications in various health conditions4. Despite the advances resulting from the extensive work performed in many labs worldwide, fundamental questions remain open regarding the assembly and organization of the MRC complexes and their impact on function and metabolic adaptation.

The MRC is organized as individual complexes that coexist with higher-order assemblies known as supercomplexes (SCs)5,6. In mammalian mitochondria, the SCs are formed by the proton-pumping enzymes−CI, CIII, and CIV−in various stoichiometries7–9. They include CI+CIII2, CIII2+CIV, and SCs CI+CIII2+CIVn, termed respirasomes because they are capable of NADH:O2 oxidoreduction in vitro. Recent in situ cryo-electron microscopy has revealed four major respirasome organizations: CI+CIII2+CIV, CI+CIII2+CIV2, CI2+CIII2+CIV, and CI2+CIII2+CIV2 10. Although the physiological roles of respirasomes remain controversial, they have been extensively characterized both structurally and biochemically10–14, particularly the simplest and most predominant form, CI+CIII2+CIV, shedding light on electron transfer, proton pumping, and regulatory mechanisms2,15.

The functional relevance of SCs may be intricately tied to the regulatory mechanisms governing their biogenesis, which are not yet fully elucidated. Two contrasting models have been proposed to explain SCs' biogenesis and their functional coexistence with individual complexes. The plasticity model posits that SCs form only after the individual complexes are fully assembled7, while the cooperative assembly model proposes that SC assembly begins with intermediates of individual complexes16,17. Emerging evidence supports the second model, showing that partially-assembled complexes can interact in the context of SCs15,18–20. Additionally, it has been hypothesized that SCs may serve as a platform for coordinated complex biogenesis regulation, influencing the assembly and stability of the individual complexes18,21,22.

Recent investigations have shown that CIV subassemblies interact with CI and CIII in SCs and have suggested that alternative assembly pathways coexist to form “free” versus “respirasome-bound” CIV19,23. CIV assembly is believed to be a modular process where the catalytic mt-COX1, mt-COX2, and mt-COX3 subunits, encoded in the mitochondrial genome, form preassembly modules with several of the eleven nucleus-encoded subunits forming the complex24,25. The maturation of each catalytic subunit and the formation of modular assemblies are assisted by more than thirty assembly factors25. The search for accessory factors assisting the assembly of CIV-containing respirasomes identified the Cytochrome c Oxidase subunit isoform 7A2-like COX7A2L26–29. While COX7A2L was initially considered essential for CIV binding in both CIII2CIV and respirasomes27,29, further structural evidence suggests it is needed for CIII2CIV formation rather than the respirasome itself30. However, complexome profiling and structural studies have identified two distinct CI+CIII2+CIV respirasome classes containing either COX7A2L or COX7A2 isoforms, preferentially assembling depending on metabolic cues31,32. On the other hand, the Hypoxia Inducible Gene Domain family protein HIGD2A binds to and promotes the association of the mt-COX3 module into both individual CIV and respirasomes23,33. Additionally, it has been proposed that HIGD2A promotes the formation of respirasome assembly intermediates by coordinating the association of CIV assembly modules, revealing divergent assembly pathways for individual and supercomplexed CIV19,34. The heterogeneity of SC populations coexisting in the inner mitochondrial membrane (IMM) and the challenge of separating SC assembly intermediates from their mature forms for structural studies has complicated assigning the roles of different factors in the SC assembly processes.

In this study, we isolated the human respirasome CI+CIII2+CIV from human embryonic kidney (HEK293) cells and solved high-resolution structures of its constituent complexes using cryo-electron microscopy (cryo-EM). We identified the late-stage respirasome assembly intermediates that capture the final steps of CIV assembly after CI and CIII are fully matured. Detailed structural and biochemical analyses revealed that HIGD2A binds to CIV, serving as a placeholder for the final incorporation of the NDUFA4 subunit, which completes CIV and finalizes respirasome biogenesis. Our findings offer critical insights into the temporal dynamics and molecular intricacies of CIV assembly, both as an isolated complex and within the context of the respirasome.

Results
Determination of human respirasome structure by cryo-EM
To gain insight into the assembly of CIV, we engineered a C-terminal FLAG tag on the CIV biogenesis factor COX14, which has previously been shown to associate with COX1 during its translation and CIV maturation within the IMM35,36. Mitochondrial membranes were solubilized with lauryl maltose neopentyl glycol (LMNG), followed by affinity purification and cryo-EM analysis of the eluates (see the “Methods” section). We detected abundant 2D classes representing unique views of the respirasome SC (Supplementary Fig 1), which were pooled for 3D reconstruction. This yielded a map of CI+CIII2+CIV respirasome with an overall resolution of 3.1 Å (Fig. 1A). The membrane domains of the complexes were visualized with a bound detergent belt displaying a slight curvature, consistent with recently reported SC structures10,37. Despite extensive data processing and map analysis, we were unable to identify any additional density corresponding to COX14, the bait protein, most likely due to its transient interaction and subsequent loss during post-immunoprecipitation processing.

In the initial map after homogeneous refinement, the density for CIV appeared weaker than CI and CIII, indicating greater conformational flexibility, in line with previous studies11–14,38. This observation was further supported by 3D variability analysis using the mask covering both CIII and CIV (Supplementary Movie 1). Local refinement on CI, CIII, and CIV significantly improved their respective resolutions to 2.9 Å, 2.77 Å, and 3.1 Å (Supplementary Fig. 1). In the following sections, we describe the structures of each complex, with a particular focus on the resolution of several CIV assembly intermediates within these SCs.

Structure of human complex I at 2.7 Å resolution
A final focus map on CI built from a total of 236,346 particles yielded 2.66 Å resolution with consistent local resolution (Supplementary Figs. 1, 2, Supplementary Table 1). We identified distinct electron densities for flavin mononucleotide (FMN) and dihydro-nicotinamide-adenine-dinucleotide phosphate (NADPH) molecules, as well as Fe–S clusters, but not for any NADH molecule (Supplementary Fig. 3A). Based on both the overall enzyme conformation and structural features of key components of CI (Supplementary Fig. 3A, B), we concluded that CI is in an active resting state, consistent with previous findings39–43. Therefore, we will not discuss the open or inactive states of the complex, as these have been thoroughly examined in earlier studies42,43 and reviewed recently44,45. Additionally, we observed ambiguous broken densities throughout the ubiquinone-binding channel (Supplementary Fig. 3A), reminiscent of those observed in previous active/closed structures of mammalian CI that also lacked a defined bound ligand39,40,43.

The improved resolution compared to earlier models of human CI14 allowed us to build a more accurate model (Supplementary Table 2). We modeled fourteen common phospholipids, including 6 cardiolipin molecules and 8 molecules of 1,2-dioleyl-sn-glycerol-3-phosphothanolamine (PEE), primarily located at the interface between CI and CIII2 (Supplementary Fig. 4A). Additionally, the nucleotide in subunit NDUFA10 was modeled as a Mg2+-bound 2’-deoxyguanosine-5’-triphosphate (Supplementary Fig. 3C, right), in agreement with recent biochemical and structural data from other species46,47. Notably, while previous studies reported the demethylation of mouse CI NDUFS2-Arg8542,47,48, our analysis revealed the corresponding human CI residue, Arg118, as monomethylated (Supplementary Fig. 3C, left). Interestingly, we found the phosphorylation at the Ser58 residue of the NDUFS7 subunit, which has not been previously reported (Supplementary Fig. 3C, middle). Taken together, this finding indicates that post-translational modification patterns may vary depending on species or tissue type.

Structure of human complex III at 2.5 Å resolution
To date, only one structure of human CIII has been resolved, with an overall resolution of 3.4 Å14. In the current study, we obtained a final focus map on CIII from a total of 213,731 particles, resulting in the CIII structure at a resolution of 2.5 Å for most regions, except for the dynamic Rieske iron-sulfur protein (RISP) (Supplementary Figs. 1, 2, Supplementary Table 1). This high-resolution map enabled us to build accurate models for most of the proteins within the complex (Supplementary Fig. 4B, C, Supplementary Table 3). CIII exists as a homodimer, in which we unambiguously modeled two metal centers per monomer, including two heme molecules (heme bL and bH) in MT-CYB and one heme c1 molecule in cytc1 (CYC1) (Supplementary Fig. 4B, insertion). Additional weak densities were observed at the two ubiquinone (UQ) binding sites, which we modeled as Q10 molecules, although we were unable to determine their redox states (Supplementary Fig. 4B, insertion). Notably, our map did not show any additional densities at the cytc binding site, which were previously reported in human respiratory megacomplex CI2CIII4CIV214. Additionally, we did not detect a clear density corresponding to the Fe-S cluster (Supplementary Fig. 4B, insertion), likely due to the conformational heterogeneity of RISP within CIII, as previously reported10,13,14.

HIGD2A- and NDUF4A-bound intermediate structures of complex IV
Local refinement of CIV revealed high flexibility, suggesting structural heterogeneity. A 3D variability analysis, using a mask generated during local refinement, revealed the presence of additional proteins within a cleft formed by COX1, COX2, COX3, and COX6A1. Interestingly, previous crystal structures of the homodimeric CIV49 suggest that this cleft plays a critical role in mediating interactions between two CIV units.

To investigate this further, we generated a mask covering the COX3 module and extending toward the cleft. As a result, the subsequent 3D classification revealed four distinct classes with varying density patterns surrounding the cleft (Fig. 1B).

Specifically, class 1 and class 4 exhibited distinct extra densities corresponding to HIGD2A and NDUFA4, respectively. Class 2 displayed density for HIGD2A, along with additional density near helix 2 of COX2, indicating the impending binding of NDUFA4 to CIV. Class 3 revealed clear extra density for NDUFA4 and a weaker density at the HIGD2A binding region, suggesting the displacement of HIGD2A upon NDUFA4 recruitment. Together, these structural analyses suggest a dynamic interplay between HIGD2A and NDUFA4 during the assembly of CIV, with HIGD2A likely being released from CIV concurrently with the incorporation of NDUFA4 (Fig. 1B).

To biochemically confirm the simultaneous presence of HIGD2A and NDUFA4 in a subset of particles, we performed FLAG-immunoprecipitation assays using wild-type (WT) HEK293T cells and HIGD2A-KO cells expressing HIGD2A-FLAG. Additionally, we performed immunoprecipitation for NDUFA4 in WT cells using anti-NDUFA4-conjugated beads. In both cases, we detected components of CI, CIII, and CIV (Fig. 1C, D), confirming that both HIGD2A and NDUFA4 interact with CIV within the context of the respirasome. Additionally, small substoichiometric amounts of NDUFA4 were co-immunoprecipitated with HIGD2A-FLAG (Fig. 1C), and similarly, small amounts of HIGD2A were co-precipitated with NDUFA4 (Fig. 1D). This observation supports the cryo-EM data, indicating that only a minor fraction of these two proteins co-exists within the same CIV assembly intermediates.

Structural features of HIGD2A-bound complex IV
Local refinement of class 1, focusing on complex IV, yielded a map at 2.93 Å resolution (Supplementary Fig. 1), exhibiting higher resolution at the core subunits and lower resolution at the periphery. An additional density corresponding to a polypeptide with 2 transmembrane helices was observed near COX3 and COX6A, but at a relatively lower local resolution in the range of 3.5 to 6 Å (Supplementary Fig. 2, class 1 of complex IV). We modeled HIGD2A into the extra density in class 1, achieving sufficient resolution to assign most amino acid side chains from Asn31 to Pro106 within the distal C-terminal domain (Fig. 2A, Supplementary Fig. 5A).

HIGD2A and its yeast functional homolog, Rcf1, belong to the HIG1 family type 2 isoforms due to their conserved (Q/I)X3(R/H)XRX3Q (QRRQ) motif (Supplementary Fig. 6A, B), which is necessary for binding COX3 and regulating modular CIV assembly23,50. The structural model revealed specific interactions of the residues Arg80, Arg82, and Glu86 of the conserved QRRQ motif, with highly conserved residues His149, Asp246, and His250 of COX3 (Fig. 2B, C). Within the IMM, HIGD2A engages in hydrophobic and Van der Waals interactions with COX3 and COX6A1 (Fig. 2D). For comparison, a previous structural study in yeast (S. cerevisiae) identified Rcf2 (PDB: 6T0B), another type 2 isoform of the HIG1 family (Supplementary Fig. 6C, middle panel), bound to the mature CIII2+CIV1–2 SCs, occupying a pocket formed by CIV subunits Cox1, Cox3, Cox12, and Cox13 (ref 51). Recently, the structure of Rcf2-bound CIV (PDB: 8C8Q52) from yeast (S. pombe) revealed specific interactions between the fully conserved Arg143 and Gln147 of the QRRQ motif and Asp254 of COX3 (Supplementary Fig. 6C, right panel). This interaction with mature CIV closely resembles that of HIGD2A, even though Rcf2 is described as modulating CIV function rather than its assembly53, and it is considered the functional homolog of human HIGD1A, a type-1 HIG1 family member (Supplementary Fig. 6A, B). Although HIGD2A’s functional homolog is Rcf1, it has been suggested that Rcf1 binds CIV in a similar manner to Rcf252, which may explain the parallels between HIGD2A binding to CIV described here and the previously characterized interaction of Rcf2 with CIV, despite their distinct functions. Nonetheless, some differences are observed between HIGD2A and Rcf2 binding to CIV. For instance, the yeast SC structure revealed the C-terminal α-helix of yeast Rcf2 extending into the IMS, to form electrostatic interactions with Cox1251. In contrast, HIGD2A lacks this C-terminal extension (Supplementary Fig. 6A), and we did not detect any additional interactions between the IMS-protruding regions of HIGD2A and COX6B1, the human homolog of yeast Cox12. Likewise, interactions observed between Rcf2 and Cox1351, which occupies a position analogous to COX6A1 in human CIV, were absent in our structural model.

In our HIGD2A-bound structure, we identified a well-defined cardiolipin (CDL) molecule with its head group inserted into a deep pocket formed by COX1, COX3, and the IMS side of COX6B1 (Supplementary Fig. 5C). This CDL likely plays a role in maintaining the structural integrity and function of the complex54. Additionally, three phosphatidylethanolamine (PEE) molecules were previously reported within COX3 in both cryo-EM and crystal structures of human CIV54. In our structure, we observed an additional lipid molecule, assigned as PEE, bound to COX3, extending toward the surface and interacting with COX7A2 (Supplementary Fig. 5E). This binding site was previously shown to be occupied by a CDL molecule in crystal structures of both dimeric55 and monomeric bovine CIV56.

Structure of NDUFA4-bound complex IV
Local refinement of class 4 led to a 3.2 Å resolution reconstruction. Overall, the map is in agreement with a previous cryo-EM structure of human CIV, resolved at 3.6 Å resolution, which identified NDUFA4 as the fourteenth subunit of CIV54 (Fig. 3A, Supplementary Fig. 1 and Supplementary Fig. 5B). Although the local resolution of the map covering the NDUFA4 region is lower than 3.2 Å, ranging from 4 to 6 Å (Supplementary Fig 2, class 4 of complex IV), clear side chain densities are visible for several bulky residues, including Phe19, Tyr32 and Trp45 (Supplementary Fig. 5B). As expected, the N- and C-termini of NDUFA4 are located on the matrix side and IMS, respectively (Fig. 3A). The N-terminal region forms a short α-helix within the IMM toward the matrix, while the C-terminus is in close contact with COX6B1 in the IMS. The central region of NDUFA4 forms a transmembrane helix (TMH) that parallels the main TMHs of COX1 and is adjacent to TMH2 of COX2 (Fig. 3A). Interestingly, the presence of NDUFA4 creates a deep pocket formed by the main TMHs of COX1 and COX3, along with the C-terminal region of COX3 and the TMH of both NDUFA4 and COX6B1 within CIV. This pocket stabilizes the binding of the CDL molecule, described above for the HIGD2A-bound complex. As a result, the density for the head of the CDL molecule is more clearly resolved in the presence of NDUFA4 compared to HIGD2A (Supplementary Fig. 5C, D).

To evaluate the functional consequences of NDUFA4 incorporation into CIV, we assessed its enzymatic activity in NDUFA4-KO cells. Consistent with previous reports57,58, the absence of NDUFA4 led to an approximately 50% reduction in CIV activity, as determined by both spectrophotometric assays (Fig. 3B) and in-gel activity staining (Fig. 4H). This reduction correlated with a comparable (~50%) decrease in endogenous respiration in NDUFA4-KO cells relative to WT HEK293T cells (Fig. 3C, D).

In agreement with our structural studies, BN-PAGE analyses revealed that NDUFA4 comigrates with other CIV subunits in both isolated CIV and CIV-containing SCs in HEK293T and 143B human cell lines (Supplementary Fig. 7A). This comigration was also observed in an NDUFB7-KO CI mutant cell line, where CIV distribution is only secondarily affected (Supplementary Fig. 7A). NDUFA4 has been proposed as the final subunit added to CIV during assembly59. To investigate this, we analyzed the distribution of NDUFA4 in assembly intermediates using BN-PAGE in 143B cybrids with homoplasmic mutations in the mtDNA-encoded CIV core subunits COX160, COX261, or an early homoplasmic mutation in COX362 (Supplementary Fig. 7B), as these subunits form assembly modules19,63. NDUFA4 was not detected in any of the CIV subassemblies (Supplementary Fig. 7C) but was only associated with the small amount of fully assembled CIV present in the COX3-mutant cybrid cell line (Supplementary Fig. 7C). This suggests that NDUFA4 is indeed incorporated during the final steps of CIV assembly.

Hierarchical incorporation of HIGD2A and NDUFA4 to CIV
Our structural data indicate that NDUFA4 and HIGD2A have partially overlapping binding sites on CIV, suggesting potential competition between these proteins for binding (Supplementary Fig. 8A). To elucidate their incorporation order, we analyzed the distribution and levels of HIGD2A- and NDUFA4-containing CIV in HIGD2A-KO and NDUFA4-KO cell lines (Fig. 4A, B, Supplementary Fig. 8B, C). In HIGD2A-KO cells, NDUFA4 levels were reduced to a similar extent as other CIV subunits. Conversely, in the absence of NDUFA4, HIGD2A redistributed from the previously described 50-kDa regulatory complex23 to fully assembled isolated or supercomplexed CIV (Fig. 4B). Quantitative analysis of HIGD2A binding to CIV in NDUFA4-KO cells was performed by excising bands corresponding to holo-CIV and SCs from a first-dimension BN-PAGE gel, followed by second-dimension SDS-PAGE and immunoblotting (Supplementary Fig. 8B). The quantification revealed that, in the absence of NDUFA4, HIGD2A accumulated in CIV and SCs more than 3-fold and 2-fold, respectively (Supplementary Fig. 8C). These findings suggest that HIGD2A is stabilized on CIV when its release is not triggered by NDUFA4.

To further investigate the potential competition between HIGD2A and NDUFA4 for CIV binding, we overexpressed each protein in NDUFA4-KO, HIGD2A-KO, and wild-type (WT) cells. In WT cells, the proteins exhibited mutual exclusivity: overexpression of HIGD2A reduced NDUFA4 binding to CIV, and vice versa (Fig. 4C, D). In HIGD2A-KO cells, NDUFA4 overexpression partially restored CIV subunit levels, assembly, and activity (Fig. 4E, F). However, overexpression of HIGD2A in both WT and NDUFA4-KO cells had detrimental effects (Fig. 4G, H). These observations support a model for the final steps of CIV assembly within the respirasome, where HIGD2A facilitates the incorporation of the COX3 assembly module23,33 and initially remains bound to CIV, acting as a placeholder. Subsequently, NDUFA4 displaces HIGD2A, completing the assembly of CIV and the respirasome assembly (Fig. 5).

Methods
Human cell lines and cell culture conditions
Human HEK293T embryonic kidney cells (CRL-3216, RRID: CVCL-0063) were purchased from ATCC. A stable human HIGD2A knock-out (KO) line in the HEK293T background was previously constructed using the TALEN technology as described23.

NDUFA4 KO human HEK293T cell pools were generated by Synthego Corporation (Redwood City, CA) by applying the CRISPR-Cas9 technology, using two guide RNAs targeted to exon 1 sequences in the NDUFA4 locus (ACGGTAAGTGGCTGTAAATG and ACTTACGCTCGGATGCTTCT). We used the pools for the clonal selection of NDUFA4 KO cell lines. Single clones were screened for NDUFA4 depletion by immunoblotting and genotyped by the PCR-amplification of the NDUFA4 locus from the genomic DNA, using the primers SeqNDUFA4-F and SeqNDUFA4-R (Supplementary Table 5), followed by the sequencing of the resulting DNA fragments (Supplementary Fig. 9).

Cybrid cell lines were constructed using enucleated control fibroblasts and the osteosarcoma 143B TK 206 rho zero cell lines as described elsewhere72. COX1 and COX2 mutant cybrid cells carry a homoplasmic mitochondrial mutation; G6930A in the COX1 gene, which generates a stop codon, and G7896A in the COX2 gene, which generates a truncated version of the protein60,61. COX3 mutant cybrid cell lines had a nearly homoplasmic frameshift mutation, 9537C ins62.

The cells were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µg/ml uridine at 37°C under 5% CO2.

Stable mammalian cell lines that enable doxycycline-inducible expression of C-terminally FLAG-tagged COX14 (COX14::FLAG) were generated using the Flp-In T-Rex human embryonic kidney 293 (HEK293) cell line (Invitrogen). The COX14 cDNA was cloned into pcDNA5/FRT/TO (Supplementary Table 6). HEK293 cells were cultured in DMEM (Dulbecco’s modified eagle medium) supplemented with 10% (v/v) FBS, 2 mM Glutamax (Gibco), and 1× penicillin/Streptomycin (Gibco). 50 µg/ml uridine, 10 μg/ml Zeocin (Invitrogen), 100 μg/ml blasticidin (Gibco) at 37°C under 5% CO2 atmosphere. Before transfection, cells were seeded in a 6-well plate and cultured in culture media devoid of selective antibiotics. Cells were transfected with the pcDNA5/FRT/TO COX14::FLAG (Supplementary Table 6) using Lipofectamine 3000 (Invitrogen) following the manufacturer’s recommendations. Fresh media supplemented with hygromycin (100 μg/ml, Invitrogen) and blasticidin (100 μg/ml) was added 48 h after transfection for positive clone selection. After two to three weeks, selected colonies were picked and cultured as single clonal populations. Cells were treated with 50 ng/ml doxycycline to confirm the inducible expression of COX14, and 24 or 48 h later, cell pellets were collected for western blot examination. The cell lines were routinely checked for mycoplasma contamination.

To rule out off-target effects during gene editing, all KO clones were reconstituted with the wild-type version of the corresponding gene. HIGD2A-Myc-DDK and NDUFA4 were cloned under the control of a CMV promoter in the pCMV6-A-Entry-Hygro plasmid (Supplementary Table 6) using Sfg1-Pme1 or Not1 sites, respectively. For transfection of the different constructs, we used LipofectamineTM 3000 Transfection Reagent (Invitrogen, Cat#L3000-015) according to the manufacturer’s instructions. Media was supplemented with 200 mg/ml of hygromycin after 24 h, and drug selection was maintained for at least 21 days.

Mitochondria isolation
Mitochondria-enriched fractions were isolated from 80% confluent 15 cm diameter plates as described73 with the modifications stated below. Briefly, cells were collected by trypsinization and homogenized with ~20 strokes of a Potter-Elvehjem homogenizer in isolation buffer (20 mM HEPES-KOH, 220 mM Mannitol, 70 mM sucrose, 1 mM EDTA and 0.5 mM PMSF). Subsequently, differential centrifugation was performed to separate the mitochondria-enriched fraction. First, a low-speed centrifugation step (800×g for 5 min) allowed the separation of intact cells, membranes and nuclei from the cytosol, and secondly, two higher speed centrifugation steps (16000×g for 15 min) were made to isolate mitochondria. Finally, the mitochondrial pellet was resuspended in sucrose buffer (10 mM HEPES, pH 7.6 and 0.5 M sucrose). Protein concentration was determined by the Lowry method.

For cryoEM experiments, isolation of mitochondria was performed as previously described in Rorbach et al74. In brief, COX14::FLAG cell lines were induced with 50 ng/ml of doxycycline for 60 h and collected at a confluency of ~90% prior to mitochondrial isolation. The cell pellet was resuspended in the cold hypotonic MSE buffer (0.6 M mannitol, 10 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.1% BSA), and ruptured on ice by a semi-automatic homogenizer (Schuett-biotech). The lysate was clarified by centrifugation at 400×g and 4°C for 10 min. The pellet was resuspended and subsequently homogenized. After 3 cycles of homogenization-centrifugation, the cell lysates were combined, and the mitochondria were pelleted by additional centrifugation at 11,000×g and 4°C for 10 min. The crude mitochondria were loaded onto the sucrose cushion (1.0 M and 1.5 M sucrose in 20 mM Tris–HCl, pH 7.4, 1 mM EDTA) and centrifuged for 1 h at 77,000×g (25,000 rpm) in a SW41 Ti rotor (Beckman Colter). The band formed by the mitochondria in the middle between 1 and 1.5 M sucrose was collected carefully and resuspended in 10 mM Tris–HCl, pH 7.4 in a 1:1 ratio, The pure mitochondrial pellet was collected after centrifugation at 11,000×g and 4°C for 15 min and then resuspended in mitochondrial freezing buffer (300 mM trehalose, 10 mM Tris–HCl pH 7.4, 10 mM KCl, 0.1% BSA, 1 mM EDTA), flash-frozen in liquid N2 and stored at −80°C.

SDS-PAGE and immunoblotting
To solubilize mitochondria in denaturing conditions, they were pelleted down and resuspended in 1X Laemmli buffer (2% SDS, 10% glycerol, 60 mM Tris-HCl, pH 6.8, 2.5% β-mercaptoethanol and bromophenol blue). 40–80 μg of mitochondrial protein extract was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, proteins were transferred to nitrocellulose membranes and incubated with specific primary antibodies listed in Supplementary Table 7. Peroxidase-conjugated anti-mouse and anti-rabbit IgGs were used as secondary antibodies (Molecular Probes). Optical densities of the immunoreactive bands were measured using the ImageJ software in digitalized X-ray film images.

Blue native electrophoresis, 2-dimension electrophoresis and In gel activity assays
To extract mitochondrial proteins in native conditions, mitochondria were pelleted and solubilized in a buffer containing 1.5 M aminocaproic acid and 50 mM Bis-Tris (pH 7.0). After optimizing solubilization conditions, we decided to use digitonin with a detergent-to-protein ratio of 4 g/g. Solubilized samples were incubated on ice for 10 min and cleared at 20,000×g for 30 min. For gel loading, the supernatant was combined with sample buffer 10X (750 mM aminocaproic acid, 50 mM Bis-Tris, 0.5 mM EDTA and 5% Serva Blue G-250).

Blue Native electrophoresis was performed as described in detail elsewhere75. Briefly, NativePAGE Novex® 3%–12% Bis-Tris Protein Gels (Life Technologies) were loaded with 60–100 μg of mitochondrial protein sample. After electrophoresis, proteins were transferred to PVDF membranes with the eBlot L1 Protein Transfer System (GenScript) and subjected to immunoblotting as indicated before.

In some of the BN gels, the bands corresponding to CIV or SC CI+CIII2+CIVn were excised from the gel and run on a second dimension SDS-PAGE after their immersion for 20 min in a denaturing solution (0.1% SDS and 0.07% β-mercaptoethanol) (Supplementary Fig. 8B).

Other gels were used for CIV activity determination by incubating them in 18 mL of phosphate buffer, pH 7.4, with 1.5 g sucrose, 10 mg 3.3’-diamidobenzidine tetrahydrochloride (DAB), 20 mg of cytochrome c, and 0.4 mg of catalase. Gel was incubated for up to 48 h at 37°C in this solution. Pictures were taken at 4, 24 and 48 h of incubation.

Immunoprecipitation
For the immunoprecipitation of HIGD2A-interacting proteins, mitochondria isolated from HEK293T cells overexpressing HIGD2A-Myc-DDK were solubilized as previously described. HIGD2A was pulled down using anti-Flag agarose beads (Sigma, Cat# A2220), and interacting proteins were eluted with 1% SDS. Similarly, NDUFA4 immunoprecipitation was performed using mitochondria isolated from wild-type HEK293T cells. An anti-NDUFA4 antibody (Supplementary Table 7) conjugated to protein A agarose beads (Thermo Scientific, Cat# 20365) was used, and interacting proteins were eluted with 1% SDS.

Measurement of mitochondrial respiratory chain complex IV activity and endogenous cell respiration
Mitochondrial cytochrome c oxidase (complex IV, CIV) activity was assessed spectrophotometrically as previously described76. Assays were performed using whole cell preparations from WT, NDUFA4-KO, and KO cells reconstituted with WT NDUFA4. Enzymatic activity was normalized to protein content and expressed relative to WT values.

Endogenous cellular respiration (CR) was measured polarographically at 37°C using a Clark-type oxygen electrode (Hansatech Instruments, Norfolk, UK)77. Trypsinized cells were washed with cold PBS and resuspended at ~3×10⁶ cells/mL in 0.5 mL of respiration buffer containing 0.3 M mannitol, 10 mM KCl, 5 mM MgCl₂, 0.5 mM EDTA, 0.5 mM EGTA, 1 mg/mL BSA, 10 mM KH₃PO₄ (pH 7.4), and 2 mM ADP freshly added. The cell suspension was immediately transferred to the polarographic chamber for measurement of endogenous CR. To stimulate maximal cytochrome c reduction, 10 mM ascorbate and 0.2 mM N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) were added in a set of experiments. In a separate series, endogenous coupled respiration was uncoupled by incremental additions of up to 0.4 μM carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP) to determine the maximal oxygen consumption rate. In both sets of experiments, CIV-dependent respiration was confirmed by inhibition with 0.8 μM potassium cyanide (KCN). All respiration rates were normalized to total cell number.

Sample preparation for cryoEM
Isolation and purification of mitochondria from a large-scale COX14::FLAG overexpressing cell line were performed as described in the above paragraph “Isolation of mitochondria”. The pure mitochondria were thawed slowly on ice, then lysed at 4°C for 40 min in a lysis buffer (25 mM HEPES-KOH, pH=7.5, 50 mM KCl, 20 mM Mg(OAc)2, 1% (v/v) LMNG, 0.1 % β-DDM, 0.01 % cardiolipin, 0.1 mM DTT, 1x cOmplete EDTA-free protease inhibitor cocktail (Roche), 40 U/μL RNAse inhibitor (Invitrogen)). The mitochondrial lysate was centrifuged at 20,000×g for 15 min at 4°C. The supernatant was then added to ANTI-FLAG M2-Agarose Affinity Gel (Sigma-Aldrich), previously equilibrated with the lysis buffer and incubated for 2 h at 4°C. After incubation, the mixture was centrifuged at 5000×g for 1 min at 4°C, the supernatant was removed, and the gel was washed three times with the wash buffers (25 mM HEPES-KOH, pH=7.5, 50 mM KCl, 20 mM Mg(OAc)2, 0.1% (v/v) LMNG (Thermo Fisher), 0.01% cardiolipin, 0.1 mM DTT). Elution was performed twice by adding the elution buffer (25 mM HEPES-KOH, pH=7.5, 50 mM KCl, 20 mM Mg(OAc)2, 0.01% (v/v) Lauryl Maltose Neopentyl Glycol (LMNG), 0.001% cardiolipin, 0.001 % glycol-diosgenin (GDN, Sigma-Aldrich), 0.1 mM DTT, 500 μg/mL 3X FLAG peptide (Sigma-Aldrich) for about 45 min at 4°C. The elution was combined, then centrifuged at 55,000 rpm in TLA55 rotor (186,000×g) for 16 h. Most of the supernatant was discarded, and the last 20 μL at the bottom was collected. Fifteen μL of elution buffer without FLAG peptide was added to resuspend the pellet by slightly pipetting. The tube was centrifuged at 20,000×g for 10 min to get rid of any insoluble part, the supernatant was then combined with 20 μL above, centrifuged one more time at 20,000×g for the last 15 min and used directly to prepare the grid. Grids were prepared using protein at a concentration of 1 mg/ml.

Cryo-EM data acquisition and data processing
Prior to cryo-EM grid preparation, grids were glow-discharged with 25 mA for 120 s using an EMS100X easiGlow glow-discharge unit. Quantifoil Cu 300 mesh (R2/2 geometry – Agar Scientific) grids covered with a thin carbon layer of ~2 nm were used for the preparation of the cryo-EM samples. Four μL of the sample was applied to the grids, incubated for 30 s, then vitrified at 4°C and 100% humidity using a Vitrobot Mk IV (Thermo Fisher Scientific) (blot 5 s, blot force 3, 595 filter paper).

All cryo-EM data collection (Supplementary Table 1) was performed with EPU (Thermo Fisher Scientific) operated on Titan Krios G3i transmission electron microscope (Thermo Fisher Scientific) at 300 kV in the Karolinska Institutet’s 3D-EM facility. Images were acquired in nanoprobe 105 kX EFTEM SA mode (0.84 Å/px) with a slit width of 20 eV using a K3 Bioquantum, for 3.5 s with 50 movie frames and a total electron dose of 35 e-/Å2. Images were collected with a targeted defocus range of −0.6 to −2 μm, with an autofocus routine at every acquisition area. A total of 17,476 movies were collected from 2 datasets.

Data processing was performed using cryoSPARC (v3.2–v4.2)78. Movie frames were aligned using Patch Motion Correction, and Contrast Transfer Function (CTF) parameters were estimated by Patch CTF correction. Particle picking was performed by automatic Gaussian blob detection (mask diameter 250–500 Å with circular blob), yielding particles that were then subjected to reference-free 2D classifications (number of classes = 200). A total of 2,508,182 particles were initially picked from two datasets. Particles were inspected, then extracted in a box size of 600 Å, and Fourier cropped to a box size of 200 Å. Iterative 2D classifications were used to select the best 2D class averages of respirasome-like particles. The ab initio reconstruction was used to create four classes. Several rounds of heterogeneous refinement were applied to obtain one of the four classes, yielding a respirasome reconstruction with high-resolution features that contains 375,422 particles. The particles were re-extracted to the full box size of 600 Å for further refinement. After homogeneous refinement of these particles, 3D classification without mask followed by iterative heterogeneous refinements was performed to get rid of classes of particles with broken densities, resulting in 336,847 particles yielding 3.1 Å resolution after homogeneous refinement (Supplementary Fig. 1). These particles were utilized for the subsequent refinement of individual complexes.

Masked refinements of the respective SC intermediates resulted in map resolutions of 2.9 Å for CI, 2.8 Å for CIII, and 3.1 Å for CIV. For CI and CIII refinement, the 3D classification using the focus mask (Supplementary Fig. 1) further discarded the subsets of particles displaying minor populations, resulting in 236,346 particles and 213,731 particles, respectively, for the final refinement. It should be noted that the unused particles exhibited lower resolution structural features or appeared to represent minor conformational states. These were not further analyzed in this study to focus on predominant structural states, as the conformational change analysis of CI and CIII has been intensively studied previously40,42–44,79,80. The resulting particles were subjected to global CTF refinement and local CTF refinement, yielding the final maps with 2.66 Å and 2.52 Å resolution (FSC 0.143) for CI and CIII, respectively (Supplementary Fig. 1, Supplementary Table 1).

For complex IV, a mask covering the COX3 module (COX3, COX5B, COX6A1 and COX7A2) and regions of NDUFA4 and HIGD2A was created. The 3D classification using this mask was performed with an output of 8 classes. These classes were investigated and combined into 4 classes representing different states (Fig. 1B, Supplementary Fig. 1). They include two major classes: class 1 contains clear extra density for HIGD2A protein, while class 4 contains clear density for NDUFA4 protein. Two other classes, class 2 and class 3, show continuous densities for both proteins, representing the intermediates. Class 2 expresses clear density for HIGD2A protein and incoming density of NDUF4A, class 3 shows clear density for NDUFA4 and weak density at HIGD2A binding region (Fig. 1B). These particles were also subjected to global and local CTF refinement, followed by local refinement, yielding the maps with a range from 2.9 to 3.6 Å resolution (Supplementary Figs. 1, 2 Supplementary Table 1). The final maps of CI, CIII, class 1 and class 4 of CIV from local refinement jobs were subjected to an autosharpen job in Phenix v1.21.181, and the output maps were used for model building.

Model building and refinement
The atomic models were built manually in COOT82. First, the previous structures of human CI (PDB: 5XTD)14, human CIII2 (PDB: 5XTE)14 and human CIV (PDB: 5Z62)54 were fitted into the corresponding map as a rigid body using Chimera X v1.683,84. Then, the fitted model was manually adjusted in COOT in a residue-by-residue manner against the density map using Ramachandran restraints, torsion restraints and appropriate map weight. Densities for lipids were manually fitted by using previously reported structures as references (PDB: 8OM147 and 5XTD14 for CI; 5XTE14 and 8UGD10 for CIII; 5Z6254 and 8UGL10 for CIV). The ligands, cofactors and modifications of proteins or subunits in individual complexes were manually inspected according to the UniProt database and experimentally determined structures reported previously (PDB: 8OM1 for CI; 8UGD for CIII; and 5Z62 for CIV). The sequence of the HIGD2A protein was built in the extra density region of class 1 of CIV with the reference model (AF-Q9BW72-F1) from the AlphaFold protein structure database85. Hydrogens were added to the models of CI and HIGD2A-bound CIV in Refmac586 of the CCP4 suite87 and to the model of CIII with Phenix.readyset88. The model was further real-space refined in Phenix v1.21.181 by subjecting it to 4–6 cycles of global energy minimization, local grid search and atomic displacement parameter (ADP) refinement. This was done using reference model restraints, with the model itself used as the reference. Additionally, Ramachandran restraints set to Oldfield (favored) and Emsley8k (allowed and outlier); and rotamer restraints targeting outliers (to fit “outliers and poor map”) were applied. Restraints for ligands were generated with Refmac586 and used for model refinement in Phenix v1.21.181.

Quantification and statistical analysis
Unless indicated, all experiments were performed at least in biological triplicate, and the results were presented as mean ± standard deviation (SD) of absolute values relative to control groups. Statistical p-values for comparison of two groups were obtained by application of a Student’s two-tailed unpaired t-test, and a p<0.05 was considered significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Summary\nIn this manuscript, the authors use cryo-EM to solve structures of the human respirasome, revealing a mixture of a fully\nassembled supercomplex bound to NDUFA4 and one featuring an immature CIV bound to HIGD2A. The structures reveal\nthat HIGD2A partially occupies the position of CIV subunit NDUFA4, concluding that HIGD2A acts as a placeholder for\nNDUFA4. Complementary biochemical analysis shows that NDUFA4 assembles late into CIV and in absence of NDUFA4,\nHIG2DA binds more stably to HIG2DA, supporting the role of HIGD2A as placeholder for the assembly of the late-stage CIV\nsubunit NDUFA4. The manuscript is relevant to the field as it adds an important piece of the puzzle with regards to the\nassembly mechanism of supercomplexes: it is in fact the first structural evidence in support of the integrated assembly\nmechanism of respirasomes, where intermediates of complexes come together in the respirasome as opposed to fully\nassembled complexes forming the respirasome.\nI believe this manuscript is of interest for a broad audience due to the implications for human metabolism and disease,\nhowever I think the authors need to address some issues before the paper can be published.\nMy main concerns are:\n1) Some data is missing, such as:\na) particle numbers indicated in the processing pipeline don’t add up and there is no explanation for what was discarded\nand why\nb) the local resolution and angular distribution plots for the final cryo-EM maps have not been reported\nc) rationale for COX14-FLAG purification when COX14 does not appear to be part of the structures is not explained\n2) The conclusions made based on the provided maps are overstated:\na) the resolution is not high enough in TMH2 of COX2 (discussed in relation to Fig 3, regarding the K-pathway) discuss\nconformational changes at the side-chain level\nb) the side-chain densities for NDUFA4 and HIGD2A are only visible for some residues, not allowing for exclusive density-\nbased subunit assignment.\nThe main problem in this regard seems to be that the overall resolution indicated in the text and in Fig S1 is way better than\nthe local resolution effectively reached in the above-mentioned regions of the structure. The issue would probably be\napparent from the local resolution plots, but these are not featured in the current manuscript. It is therefore crucial that the\nauthors provide the missing information and revise the results section to avoid overinterpreting the data.\n3) The functional relevance of HIGD2A as a placeholder for NDUFA4 is unclear.\nMore details on my main concerns, as well as some minor comments, are included in the text below.\nDetailed comments\nIn line 36, “SC assembly” should be replaced by “the canonical respirasome assembly”, as the findings of the paper only\npertain to this supercomplex.\nLines 40 to 43: unclear. Which binding factors are the authors referring to here? None seems to be mentioned in the\nmanuscript.\nLine 54: the reducing equivalents that fuel the MRC are contained in NADH and succinate, not NADH and FADH2. FADH2\nis a cofactor of CII, analogous to FMN in CI, while NADH and succinate are the reduced species that donate electrons to CI\nand CII, respectively.\nLines 128-129: where is COX14 in the structures? Since it is used for affinity purification, it would be expected to find it in the\nstructures of the purified material. It appears that COX14 is an assembly factor involved in the biogenesis of the COX1\nmodule, so it is not clear how it would be bound to/allow purification of late assembly intermediates and fully assembled\nsupercomplexes. The authors should clarify this aspect. It is possible that the observed structures derive from the extremely\nabundant respirasomes present in the mitochondrial membranes, but this would mean the structures come from\n“contaminants” of the purification and this should be stated clearly. The current wording is misleading, as it suggests that\ntagging COX14 was necessary to purify the solved structures. If instead this was indeed the case, the authors should explain\nhow COX14 would allow the purification of fully assembled and late assembly respirasomes.\nLines 139-140: where are the results of 3D variability analysis of CIV shown?\nLines 248-251: based on the provided map (HIGD2A_bound_complexIV_map.ccp4), it does not appear that most side\nchains from residue 31 to 106 show unambiguous density. As commented below for figure S1, it appears that the reported\nresolutions don’t match the quality of the maps and the peripheral region of CIV, where HIGD2A is located, is strongly\naffected by the issue. While there is density for some bulky residues (such as Phe39, Tyr62, Arg80) and the overall topology\nis in good agreement with the alphafold prediction provided by UniProt\n(https://www.uniprot.org/uniprotkb/Q9BW72/entry#structure), which point towards the density being indeed HIGD2A, “with\nsufficient resolution to unambiguously resolve most amino acid side chains from Asn31 to Pro106 at the distal C-terminal\ndomain” is an overstatement.\nLines, 302-304: as above, the resolution of the map region corresponding to NDUFA4 appears to be worse than 3.2 A.\nAlthough the agreement with the previously reported structure (PDB 5Z62) and the good side chain density for some bulky\nresidues (such as Phe19 or Tyr 32) support the density assignment, which is hereby not disputed, the wording in the text is\nmisleading. I suggest removing this sentence.\nLines 258-270: it would be good to add to Fig S5 a comparison of the CIV structures from yeast CIII2CIV/CIII2CIV2 featuring\nRcf and the human CIV featuring HIGD2A from this study, to make it easier to follow the paragraph. Also, in line 263 the\nauthors refer to Rcf1, while in the rest of the paragraph they refer to Rcf2, so Rcf1 appears to be a mistake.\nLines 332-334: based on the provided maps, the whole TMH2 of COX2 does not show very high-resolution density,\ntherefore making side chain-based conclusions (showed in Fig 3C and S4 F-G) appears to be an overstatement. What\nappears to be data-supported is that NDUFA4 stabilizes TMH2 of COX2 compared to the HIGD2A-featuring CIV, but if the\nrationale provided in lines 327-328 is followed and higher flexibility is necessary for proton uptake, it is then unclear why an\nassembly intermediate (CIV featuring HIGD2A) would be more “proton uptake ready” than the fully assembled complex (CIV\nfeaturing NDUFA4). Related to this, the discussion (lines 459-463) should be edited accordingly.\nLine 338: a comment remained in the text\nLines 338-339: Ref 53 already showed that NDUFA4 is a subunit of CIV, so while it is true that this study confirms the fact, I\nsuggest the previous paper be cited here.\nLine 340: the authors do not provide any evidence, apart from the structure-based speculation, that NDUFA4 regulates\nproton transfer by stabilizing the entry point of the K-pathway. I suggest rephrasing to “NDUFA4 binding might regulate\nproton transfer”, or providing biochemical evidence and/or references from other studies that this is indeed the case.\nLines 315-341: based on my comments on lines 332-334 and 340, I suggest the authors revise this section, alongside Figs 3\nand S4, by (1) limiting the discussion to secondary structure-level conformational changes of COX2 TMH2, which are well\nsupported by the data and (2) clarifying the implications of a NDUFA4-mediated stabilization of COX2 TMH2 for the proton\ntransfer.\nFigure 4D, there is no signal for HIGD2A in the WT in the respirasome area of the gel, making it difficult to conclude that\noverexpression of NDUFA4 reduces HIGD2A binding to CIV (lines 393-397), with relation to the respirasome assembly cited\nin line 404. Also, in Figure 4F there seems to be less NDUFA4 in the respirasome of HIGD2A-KO+NDUFA4 than in\nHIGD2A-KO+EV, but looking at the level of NDUFA4 in monomeric CIV or the ATP5A loading control it does not seem like\nthe difference would be ascribed to loading different amount of material: what would be the explanation for this?\nLines 417-423: there are no bar graphs in the figure, but indeed it would be good to see them.\nLines 459-463: can the authors point to activity measurements proving that in absence of NDUFA4 oxygen reduction is less\nefficient?\nLines 464-466: how is the data helping understand the underlying pathogenic mechanisms of Leigh syndrome? What kind of\ninformation are the current structures adding to the state of the art? It appears that references 68 and 69 concern patients in\nwhich NDUFA4 is absent, or almost absent, so how are the presented data helping to explain the pathogenic mechanisms\nof Leigh syndrome?\nFigure S1: The particle numbers don’t add up. The consensus refinement indicates 301k particles, from which the local\nrefinements are performed. But the total of the four CIV classes is around 334k particles, so it is not clear where the\nadditional particles come from.\nAdditionally, it appears that only a subset of the particles were used to refine CI (168k particles) and CIII (213k particles). In\nthe Methods section, the authors state that “the focus mask further removed the poor alignment particles”, but this does not\nseem very likely because in the respirasome CI and CIII usually drive the alignment, partly because the membrane arm of CI\nand the proximal protomer of CIII form the “rigid core” of the respirasome and also because the eight FeS clusters of CI and\nthe six hemes of CIII provide strong densities that aid the particle alignment. It is therefore strange that CI and CIII would be\npoorly aligned in the respirasome, particularly CI which “loses” almost half of the particles from the consensus refinement to\nthe focused refinement. As there is no data shown for the processing steps leading to the final maps of CI and CIII, one\ncannot see how the removed classes look like, or at which stages they were removed. It is therefore important that the\nauthors show exactly how the data was processed for CI and CIII. Since so many particles are removed, is it possible that\nother intermediates, perhaps lacking the N-module of CI, are present in the dataset and were discarded as “poorly aligned”?\nOr are the discarded particles corresponding to the open state of CI? Also, what is the overlap between discarded particles\nfor CI and CIII? Meaning, are the discarded particles coming from the same respirasome particles? If so, is it possible that\nthose are generally damaged particles, which therefore should not be considered for CIV refinement either? When focused\nrefinements are run starting from a consensus map, to boost the resolution, one would expect that the number of particles\ndoes not change because this is the pool that corresponds to the structure of interest, in this case the respirasome.\nRemoving one third (as was done for CIII) or half of the particles (as was done for CI) is unexpected and appears unjustified,\ntherefore needs to be clarified and supported by data.\nLines 676-678: Along the same line, in the methods section it is stated that the CIV focused classification was done with 6\nclasses, then the 6 classes should be shown in the supplementary figure, also indicating which classes were pooled based\non their overlapping features.\nFigure S1 lacks:\n1) the map-to-model FSC curves for each modelled structure\n2) the half-map FSC curve for the consensus map\n3) the local resolution depictions (i.e. surface colored by local resolution), as well as\n4) the angular distribution plots for each focused map and the consensus map.\nThe description of how the plots and colored maps are obtained should then also be added to the methods section. The\nmethods section should also state whether the maps were sharpened and if so how.\nPoints 3 and 4 are particularly important because the provided maps don’t look like they correspond to the reported\nresolutions, so the local resolution coloring would clarify which regions of the maps really reach the reported resolution and\nthe angular distribution would shed light on a potential anisotropy issue, which might contribute to explaining why the maps\ndon’t look like they correspond to the reported resolution.\nAs the “class 2” and “class 3” complex IV maps provided don’t look anisotropic, it might also be that the weird appearance of\nthe ccp4 maps is a sharpening artifact, which is why it would be important to declare whether the maps were sharpened.\nMinor comments\nFigure S4: in panels F and G, the subunit to which Y244 and K319 belong (COX1 presumably) should be indicated in the\nlegend.\nAll figures displaying densities (Figs. 1, S2, S3, S4) should indicate the contour used in the legend.\nLine 604: the section is titled “immunoprecipitation”, but lines 606-628 feature the description of sample purification for cryo-\nEM. I suggest this section is thus separated and titled “cryo-EM sample preparation”\nLegend to Fig S2: state that NDP stands for NADPH, as this is not clarified.\nFig 2D: the figure, or legend, lack reference to the scalebar used to calculate the electrostatic potential.", "reviewer_2": "The assembly of the respiratory chain and supercomplex formation plays important roles in human health and disease. This\npaper describes a cryo-EM study of human respirasomes, purified using an engineered tag on a complex IV subunit. The\nanalysis revealed I2III2IV1 supercomplexes with high-resolution structures of fully assembled CI and CIII, while CIV density\nis weak. Local refinement and classification of the flexibly attached CIV yielded distinct classes, containing either the\nassembly factor HIGD2A and/or the subunit NDUFA4, which is the last subunit to be added to the complex.\nIn this interesting paper the authors make a convincing case that the removal of HIGD2A is necessary for insertion of\nNDUFA4, as their positions partially overlap. However, the authors do not mention what, if any, role HIGD2A plays in\nrespirasome formation. Also, it is not clear if acting as a “placeholder” for NDUFA4 is the only function of HIGD2A or if it\nplays a role in the attachment of the COX3 module, as previously suggested. The paper would be strengthened if these\nissues were addressed.\nSpecific comments:\n1. The protein was purified with a tag on CIV, so presumably yielding both respirasomes and single CIV. However, the latter\nis never mentioned. Are the particles visible in the micrographs? If so, why are these not analysed? If not, how can this be\nexplained?\n2. The introduction, p. 4 states “it has been proposed that HIGD2A promotes the formation of respirasome assembly\nintermediates by coordinating the association of CIV assembly modules, revealing divergent assembly pathways for\nindividual and supercomplexed CIV”. Is there any evidence for this? For a distinction it would be necessary to study\nindividual CIV as well. It is nowhere shown how CIV is oriented in the respirasome and what subunits interact with CI or\nCIII.\n3. Further to that, the model in Fig. 5 does not show what induces CIV attachment to the respirasome.\n4. p. 12: The story about the flexibility at the K-pathway entrance is confusing. The region is said to typically have high B\nfactors, indicating flexibility, nevertheless distinct conformations are assigned to these uncertain densities. Particularly\nsuspicious is Glu62: a negatively charged side chain typically has no cryo-EM density (unless the charge is compensated by\na nearby positive charge like a salt bridge or ion ligand) and the side chain can't be modeled correctly even in a rigid\nstructure. The absence of the glutamate sidechain is obvious in Suppl. Fig. 4F and G (although just showing density for one\nresidue does not give an indication of the level of flexibility in this region). Thus, the suggestion that the conformation of\nGlu62 in a bovine CIV structure in GDN represents an intermediate state between the two human structures is a very weak\nargument (Fig. 3C). If the authors feel that there is a real conformational change in this region, better figures will be needed\nshowing the actual densities, not just models. Otherwise, this section needs to be toned down. Further to suppl. fig. 4F,G:\nplease indicate which subunits the highlighted residues belong to. Glu62 is misspelled as Glu61 in the legend.\n5. Figure 1B: please indicate how CIV is oriented relative to the respirasome shown in 1A.\n6. Cryo-EM methods (p.23):\na. What is the protein concentration?\nb. What microscope was used?\nc. Please add “total electron dose” of 35 e-/Å2.\nd. How many particles were picked?\ne. “bad alignment particles” and “poor alignment particles”: please rephrase.\nf. What is a “local refinement volume”?\ng. Global or local CTF refinement?\n7. Model building (p. 24):\na. Densities for lipids were manually fitted using references: please explain.\nb. “The ligands, cofactors and modifications were manually inspected as suggested in Uniprot and experimentally\ndetermined structures reported previously”. Unclear.\nc. HIGD2A was manually built with an AlphaFold structure as reference. Please remove “manually”.\n8. Suppl. fig. 1: The workflow needs more details/images: exemplary micrographs, 2D class averages, number of particles\npicked initially, initial models."}, "author_response": "Response to reviewers’ comments\nWe thank the reviewers for their time and effort in evaluating our manuscript and for raising\ninsightful questions. Their detailed and thoughtful feedback has been instrumental in enhancing\nthe quality of our work.\nReviewer #1 (Remarks to the Author):\nSummary\nIn this manuscript, the authors use cryo-EM to solve structures of the human respirasome,\nrevealing a mixture of a fully assembled supercomplex bound to NDUFA4 and one featuring\nan immature CIV bound to HIGD2A. The structures reveal that HIGD2A partially occupies\nthe position of CIV subunit NDUFA4, concluding that HIGD2A acts as a placeholder for\nNDUFA4. Complementary biochemical analysis shows that NDUFA4 assembles late into\nCIV and in absence of NDUFA4, HIG2DA binds more stably to HIG2DA, supporting the\nrole of HIGD2A as placeholder for the assembly of the late-stage CIV subunit NDUFA4. The\nmanuscript is relevant to the field as it adds an important piece of the puzzle with regards to\nthe assembly mechanism of supercomplexes: it is in fact the first structural evidence in\nsupport of the integrated assembly mechanism of respirasomes, where intermediates of\ncomplexes come together in the respirasome as opposed to fully assembled complexes\nforming the respirasome.\nI believe this manuscript is of interest for a broad audience due to the implications for human\nmetabolism and disease, however I think the authors need to address some issues before the\npaper can be published.\nWe would like to thank the reviewer for their positive evaluation and thorough analysis of the\nmanuscript. This careful set of comments is greatly appreciated.\nMy main concerns are:\n1) Some data is missing, such as:\na) particle numbers indicated in the processing pipeline don’t add up and there is no\nexplanation for what was discarded and why\nWe have revised Supplementary Figure 1 to provide additional details on data processing,\nincluding updated particle counts and an explicit indication of discarded particles.\nCorresponding updates have also been made to the Data Processing section of the Methods.\nLines 732-755:\nA total of 2,508,182 particles were initially picked from two data sets. Particles were inspected,\nthen extracted in a box size of 600 Å and Fourier cropped to a box size of 200 Å. Iterative 2D\nclassifications were used to select the best 2D class averages of respirasome-like particles. The\nab initio reconstruction was used to create four classes. Several rounds of heterogeneous\nrefinement were applied to obtain one of the four classes, yielding a respirasome reconstruction\nwith high resolution features (375,422 particles). The particles were re-extracted to the full box\nsize of 600 Å for further refinement. After homogeneous refinement of these particles, 3D\nclassification without mask followed by iterative heterogeneous refinements was performed to\nget rid of classes of particles with broken densities, resulting in 336,847 particles yielding 3.1\nÅ resolution after homogeneous refinement (Supplementary Figure 1). These particles were\nutilized for the subsequent refinement of individual complexes.\nMasked refinements of the respective SC intermediates resulted in map resolutions of 2.9 Å\nfor CI, 2.8 Å for CIII, and 3.1 Å for CIV. For CI and CIII refinement, the 3D classification\n1\nusing the focus mask (Supplementary Figure 1) further discarded the subsets of particles\ndisplaying minor populations, resulting in 236,346 particles and 213,731 particles,\nrespectively, for the final refinement. It should be noted that the unused particles exhibited\nlower resolution structural features or appeared to represent minor conformational states. These\nwere not further analyzed in this study to focus on predominant structural states, as the\nconformational change analysis of CI and CIII has been intensively studied previously 1-6. The\nresulting particles were subjected to global CTF refinement and local CTF refinement, yielding\nthe final maps with 2.66 Å and 2.52 Å resolution (FCS 0.143) for CI and CIII, respectively.\nb) the local resolution and angular distribution plots for the final cryo-EM maps have not been\nreported\nWe have added a new Supplementary Figure 2, which includes local resolution and angular\ndistribution for all reported maps.\nc) rationale for COX14-FLAG purification when COX14 does not appear to be part of the\nstructures is not explained\nWe employed COX14-FLAG purification to capture assembly intermediates of CIV (as\ndescribed previously by Richter-Dennerlein et al, doi: 10.1016/j.cell.2016.09.003).\nUnexpectedly, the most prominent and stable complexes obtained through our procedure, and\nsubsequently visualized by cryo-EM, were supercomplexes, whereas COX14 itself was not\ndetected in the final structures. Notably, our immunoprecipitation analyses, together with a\ncloser examination of the mass spectrometry data reported by Richter-Dennerlein et al., support\nthe presence of the supercomplexes interacting with COX14. As this finding has been observed\nacross different laboratories, it is unlikely to represent an artifact of our procedure. Instead, it\nsuggests that the insertion of mitochondrially encoded proteins into CIV occurs in the context\nof supercomplexes—an observation we emphasize throughout the manuscript.\nReciprocal IP analyses using HIGD2A and NDUFA4 as baits (Figure 1) also detected COX14,\nalthough only a small fraction of COX14 was recovered in the eluates, suggesting a transient\ninteraction with the captured complexes. In our experience, it is common for bait proteins to\nbe partially or fully lost during purification, particularly when the interaction is weak or\ntransient. Similar challenges have been reported by others: for example, Lenaric et al.\nhttps://doi.org/10.1038/s41467-021-23811-8 used MTG1-FLAG to purify the mitoribosome\nbut did not visualize MTG1 in any observed mitoribosomal large subunit assembly states, while\nCheng et al. https://doi.org/10.1038/s41467-021-24818-x reported that GTPBP10 was present\nin only ~1% of mitoribosomal complexes despite being used as bait.\nIn the case of our COX14 IP, additional post-immunoprecipitation centrifugation steps likely\ncontributed to its loss. Specifically, we performed a 16-hour centrifugation at 55,000 rpm in a\nTLA55 rotor to enrich for higher-molecular-weight complexes and minimize interference from\nsmaller complexes in downstream cryo-EM analyses (see Sample preparation for cryo-EM in\nthe Methods section).\nAs the mechanistic basis of COX14 interactions turned out not to be the primary focus of this\nstudy, it remains to be explored in future work. Nonetheless, we have clarified in the main text\nthat COX14 was not detected in the structural analysis, likely due to its transient interaction\nand subsequent loss during post-IP preparations.\nWe added this sentence to clarify in the manuscript\nTo gain insight into the assembly of CIV, we engineered a C-terminal FLAG tag on the CIV\nbiogenesis factor COX14, which has previously been shown to associate with COX1 during its\ntranslation and CIV maturation within the IMM 7,8.\nLine 140-143:\nDespite extensive data processing and map analysis, we were unable to identify any additional\ndensity corresponding to COX14, the bait protein, most likely due to its transient interaction\nand subsequent loss during post-immunoprecipitation processing.\n2) The conclusions made based on the provided maps are overstated:\na) the resolution is not high enough in TMH2 of COX2 (discussed in relation to Fig 3, regarding\nthe K-pathway) discuss conformational changes at the side-chain level\nb) the side-chain densities for NDUFA4 and HIGD2A are only visible for some residues, not\nallowing for exclusive density-based subunit assignment.\nThe main problem in this regard seems to be that the overall resolution indicated in the text and\nin Fig S1 is way better than the local resolution effectively reached in the above-mentioned\nregions of the structure. The issue would probably be apparent from the local resolution plots,\nbut these are not featured in the current manuscript. It is therefore crucial that the authors\nprovide the missing information and revise the results section to avoid overinterpreting the\ndata.\nThank you for pointing this out. We agree that the local resolution in peripheral regions,\nincluding NDUFA4 and HIGD2A, is lower than the overall resolution indicated in the text and\nSupplementary Figure 1. We have now included maps showing local resolution in\nSupplementary Figure 2 and revised the text accordingly. In addition, we have removed the\nword ‘unambiguous.’\nLine 257-260:\nAn additional density corresponding to a polypeptide with 2 transmembrane helices was\nobserved near COX3 and COX6A, but at a relatively lower local resolution in the range of 3.5\nto 6 Å (Supplementary Figure 2, class 1 of complex IV).\nAlthough the resolution of TMH2 of COX2 is also lower than GSFSC resolution, the electron\ndensity for Glu62 residue in NDUFA4-bound complex IV is better visualized than that in\nHIGD2A-bound complex, as shown in Supplementary Figure 5F and 5G (Supplementary\nFigure 4 in the previous version). Furthermore, we added Supplementary Figure 5H and 5I to\ndisplay the B factor of the COX2 subunit in both structures. While the B-factor ranges are\nalmost similar for the two models, the region of the K-pathway entrance (Glu62 as a\nrepresentative residue) in the HIGD2A-bound state displays a greater B-factor than that in the\nNDUFA4-bound state. Therefore, we concluded that the region in the HIGD2A-bound\ncomplex IV may be more flexible than in the NDUFA4-bound complex. However, as the\nfunctional consequences of this difference remain unclear, we chose to moderate our\ninterpretation. Accordingly, we revised the text and removed the concluding sentence from the\nResults section.\nThe density for Glu62 of COX2 in the HIGD2A-bound CIV suggests uncertainty regarding\nside chain conformation, consistent with high flexibility (Supplementary Figure 4F, H).\nInterestingly, this region appears slightly more ordered in the presence of NDUFA4\n(Supplementary Figure 5G; I), indicating a potential stabilizing effect.\n3) The functional relevance of HIGD2A as a placeholder for NDUFA4 is unclear.\nThe primary previously studied function of HIGD2A is its involvement in COX3 module\nbiogenesis 9-11.\nIn the Discussion, we speculate that HIGD2A may act as a placeholder to maintain the complex\nin a partially assembled state until the appropriate NDUFA4 isoform is incorporated. This\nhypothesis is supported by the structural and biochemical evidence suggesting that HIGD2A\nand NDUFA4 are inserted consecutively.\nInterestingly, both HIGD2A and the NDUFA4L2 isoform are known to be regulated by oxygen\nlevels. This raises the possibility that HIGD2A plays a regulatory role in adapting complex IV\nassembly to oxygen availability.\nWe acknowledge that this proposed new function warrants further investigation, particularly\nunder defined oxygen conditions and with isoform-specific analyses.\nWe modified the text to highlight that the functional relevance needs to be further investigated.\nLine 505-508:\nFurther investigation will be required to explore the mechanistic link between either NDUFA4\nor HIGD2A incorporation, K-pathway stabilization, and efficient oxygen reduction in both\nisolated CIV and the respirasome.\nMore details on my main concerns, as well as some minor comments, are included in the text\nbelow.\nDetailed comments\nIn line 36, “SC assembly” should be replaced by “the canonical respirasome assembly”,\nas the findings of the paper only pertain to this supercomplex.\nWe changed it. Thank you for pointing it out.\nLine 36-38:\nThese structures, along with biochemical data, reveal that the canonical respirasome assembly\nconcludes with the final maturation of CIV in association with mature CI and CIII\n2.\nLines 40 to 43: unclear. Which binding factors are the authors referring to here? None seems\nto be mentioned in the manuscript.\nIt refers in this case to the different isoforms of NDUFA4, as we mention in the Discussion.\nFor clarity, we have now replaced the sentence with:\nLine 41-45:\nplaceholders like HIGD2A prevent premature recruitment of the final components of the\ncomplexes, NDUFA4 or its isoforms, and act as molecular clocks to ensure the correct\nprogression of pre-SC particles into fully functional respirasomes.\nLine 54: the reducing equivalents that fuel the MRC are contained in NADH and succinate, not\nNADH and FADH2. FADH2 is a cofactor of CII, analogous to FMN in CI, while NADH and\nsuccinate are the reduced species that donate electrons to CI and CII, respectively.\nWe changed the text to clarify.\nLine 53-56:\nThe mitochondrial respiratory chain (MRC) consists of four multimeric enzymatic complexes\n(CI to CIV) and two mobile electron carriers, ubiquinone (Q) and cytochrome c (cyt c), which\nact in concert to drive the transfer of electrons from substrates such as NADH and succinate,\nto molecular oxygen.\nLines 128-129: where is COX14 in the structures? Since it is used for affinity purification, it\nwould be expected to find it in the structures of the purified material. It appears that COX14 is\nan assembly factor involved in the biogenesis of the COX1 module, so it is not clear how it\nwould be bound to/allow purification of late assembly intermediates and fully assembled\nsupercomplexes. The authors should clarify this aspect. It is possible that the observed\nstructures derive from the extremely abundant respirasomes present in the mitochondrial\nmembranes, but this would mean the structures come from “contaminants” of the purification\nand this should be stated clearly. The current wording is misleading, as it suggests that\ntagging COX14 was necessary to purify the solved structures. If instead this was indeed the case,\nthe authors should explain how COX14 would allow the purification of fully assembled and late\nassembly respirasomes.\nAs explained in our response to the previous comment (1c), we have revised the text to avoid\nconfusion. We would like to emphasize that reciprocal IP analyses using HIGD2A and\nNDUFA4 as baits (Figure 1) detected COX14, albeit only in small amounts, suggesting that\nthe protein is still present during the late stages of respirasome assembly.\nLines 139-140: where are the results of 3D variability analysis of CIV shown?\nWe now added a Supplementary Video 1 to show the movement of Complex IV relative to\nComplex I and III. Briefly, to prove the conformational movement of Complex IV, we fixed\nthe Complex I position and used the mask covering Complex III and Complex IV for 3D\nvariability analysis (cryoSPARC). The 3D variability display job was used to create a volume\nseries of 20 intermediate reconstructions that express the conformational variability of\nComplex III and Complex IV relative to Complex I. While the relative position of Complex III\nis almost stable, Complex IV adopts a variable conformation. This observation indicated the\nconformational flexibility of Complex IV.\nLines 248-251: based on the provided map (HIGD2A_bound_complexIV_map.ccp4), it does\nnot appear that most side chains from residue 31 to 106 show unambiguous density. As\ncommented below for figure S1, it appears that the reported resolutions don’t match the quality\nof the maps and the peripheral region of CIV, where HIGD2A is located, is strongly affected\nby the issue. While there is density for some bulky residues (such as Phe39, Tyr62, Arg80) and\nthe overall topology is in good agreement with the alphafold prediction provided by UniProt\n(https://www.uniprot.org/uniprotkb/Q9BW72/entry#structure), which point towards the\ndensity being indeed HIGD2A, “with sufficient resolution to unambiguously resolve most\namino acid side chains from Asn31 to Pro106 at the distal C-terminal domain” is an\noverstatement.\nAs mentioned in the previous comment, we agree that the local resolutions at peripheral regions\nincluding NDUFA4 and HIGD2A are lower than overall resolution indicated in the text and\nSupplementary Figure. We now reported the maps showing local resolutions in Supplementary\nFigure 2 and changed the text accordingly. We also removed word “unambiguous”. (Lines 260-\n262)\nLines, 302-304: as above, the resolution of the map region corresponding to NDUFA4 appears\nto be worse than 3.2 A. Although the agreement with the previously reported structure (PDB\n5Z62) and the good side chain density for some bulky residues (such as Phe19 or Tyr 32) support\nthe density assignment, which is hereby not disputed, the wording in the text is\nmisleading. I suggest removing this sentence.\nThank you for raising this point. We rephrased this sentence as below:\nLine 325-328:\nAlthough the local resolution of the map covering the NDUFA4 region is lower than 3.2 Å,\nranging from 4 to 6 Å (Supplementary Figure 2, class 4 of complex IV), clear side chain\ndensities are visible for several bulky residues, including Phe19, Tyr32 and Trp45.\nLines 258-270: it would be good to add to Fig S5 a comparison of the CIV structures from\nyeast CIII2CIV/CIII2CIV2 featuring Rcf and the human CIV featuring HIGD2A from this\nstudy, to make it easier to follow the paragraph. Also, in line 263 the authors refer to Rcf1,\nwhile in the rest of the paragraph they refer to Rcf2, so Rcf1 appears to be a mistake.\nWe thank the reviewer for this helpful suggestion. The mention of Rcf1 in previous line 263\nwas correct. While the nomenclature of HIG domain-containing proteins can be confusing, our\nintent was not to imply strict homology between yeast and human proteins. Rather, Rcf1 and\nHIGD2A appear to fulfill functionally similar roles in the assembly of Complex IV, particularly\nin stabilizing early intermediates involving the COX3 module, as shown in recent studies 9,10.\nBy contrast, yeast Rcf2 functions later in respiratory chain biogenesis, associating with mature\nCIV and CIII₂–CIV supercomplexes, where it regulates CIV activity rather than promoting its\nassembly. This regulatory role of Rcf2 therefore seems more analogous to that of human\nHIGD1A. Nevertheless, sequence analyses indicate that both Rcf1 and Rcf2 share similarity\nwith HIGD2A, especially through the conserved QRRQ motif (Supplementary Figure 6B).\nStructural comparisons between Rcf2-bound yeast SC (PDB: 6T0B) and CIV (PDB: 8C8Q)\n(Supplementary Figure 6C) reveal similar binding positions. However, these similarities do not\nnecessarily imply overlapping functional roles for these proteins in mitochondrial respiratory\nchain organization.\nWe have modified the text to clarify this:\nLine 276-284:\nThis interaction with mature CIV closely resembles that of HIGD2A, even though Rcf2 is\ndescribed as modulating CIV function rather than its assembly 12, and it is considered the\n6\nfunctional homolog of human HIGD1A, a type-1 HIG1 family member (Supplementary Figure\n6A-B). Although HIGD2A’s functional homolog is Rcf1, it has been suggested that Rcf1 binds\nCIV in a similar manner than Rcf2 13, which may explain the parallels between HIGD2A\nbinding to CIV described here and the previously characterized interaction of Rcf2 with CIV,\ndespite their distinct functions. Nonetheless, some differences are observed between HIGD2A\nand Rcf2 binding to CIV…\nLines 332-334: based on the provided maps, the whole TMH2 of COX2 does not show very\nhigh-resolution density, therefore making side chain-based conclusions (showed in Fig 3C and\nS4 F-G) appears to be an overstatement. What appears to be data-supported is that NDUFA4\nstabilizes TMH2 of COX2 compared to the HIGD2A-featuring CIV, but if the rationale\nprovided in lines 327-328 is followed and higher flexibility is necessary for proton uptake, it\nis then unclear why an assembly intermediate (CIV featuring HIGD2A) would be more “proton\nuptake ready” than the fully assembled complex (CIV featuring NDUFA4). Related to this,\nthe discussion (lines 459-463) should be edited accordingly.\nAs mentioned above, we have updated Supplementary Figure 5 to include panels S5H and S5I,\nwhich present the B-factors of the COX2 protein in both HIGD2A- and NDUFA4-bound\ncomplex IV. We agree with the reviewer that, while we wish to report these observations, it\nwould not be appropriate to base strong claims on subtle structural differences. As this evidence\nis not particularly strong, we have therefore moderated the results section (as mentioned above)\nand our discussion.\nLine 501-508\nOur structures suggest the possibility that NDUFA4 binding may influence proton transfer by\nstabilizing the entry point of the K-pathway. In this scenario, oxygen reduction at the CIV\ncatalytic center may proceed efficiently only in the fully mature holoenzyme, possibly linking\nNDUFA4 binding to the functional activation of CIV, both on its own and within the\nrespirasome. Further investigation will be required to explore the mechanistic link between\neither NDUFA4 or HIGD2A incorporation, K-pathway stabilization, and efficient oxygen\nreduction in both isolated CIV and the respirasome.\nLine 338: a comment remained in the text\nRemoved.\nLines 338-339: Ref 53 already showed that NDUFA4 is a subunit of CIV, so while it is true\nthat this study confirms the fact, I suggest the previous paper be cited here.\nAdded.\nLine 340: the authors do not provide any evidence, apart from the structure-based speculation,\nthat NDUFA4 regulates proton transfer by stabilizing the entry point of the K-pathway. I\nsuggest rephrasing to “NDUFA4 binding might regulate proton transfer”, or providing\nbiochemical evidence and/or references from other studies that this is indeed the case.\nLines 315-341: based on my comments on lines 332-334 and 340, I suggest the authors revise\nthis section, alongside Figs 3 and S4, by (1) limiting the discussion to secondary structure-level\nconformational changes of COX2 TMH2, which are well supported by the data and (2)\nclarifying the implications of a NDUFA4-mediated stabilization of COX2 TMH2 for the proton\ntransfer.\nmentioned above).\nFigure 4D, there is no signal for HIGD2A in the WT in the respirasome area of the gel,\nmaking it difficult to conclude that overexpression of NDUFA4 reduces HIGD2A binding to\nCIV (lines 393-397), with relation to the respirasome assembly cited in line 404.\nWe thank the reviewer for indicating this point. Detecting a strong HIGD2A signal in\nsupercomplexes (SCs) by BN-PAGE is technically difficult, as this protein is present at low\nlevels in these structures. Considering that only ~30% of total CIV in the mitochondrial\nrespiratory chain is incorporated into SC I+III₂+IV14 (DOI: 10.1074/jbc.M106474200), and\nonly a fraction of that has HIGD2A bound, detecting or quantifying the effect of NDUFA4\noverexpression on this specific structure by BN-PAGE is particularly challenging. We often\nneed to overexpose the film to begin seeing respirasome-bound HIGD2A, which results in high\nbackground and overly intense signals in other bands, limiting interpretability. Nevertheless,\nwe were able to detect the respirasome-bound HIGD2A in two of the three experiments we\nperformed (Figure R1), although these images are not of enough quality to be published.\nImportantly, HIGD2A stabilization in the absence of NDUFA4 has been observed\nboth in isolated CIV and in respirasome-bound CIV. Therefore, although the displacement of\nHIGD2A upon NDUFA4 overexpression is only biochemically evident in isolated CIV, we\nhypothesize that the same mechanism may also occur within the supercomplex.\nFigure R1: Distribution of HIGD2A in CIV and CIV-containing supercomplexes analyzed by BN-\nPAGE of isolated mitochondria from WT cells overexpressing HIGD2A (2A), NDUFA4 (A4), or carrying\nan empty vector (EV) in two independent experiments. Green arrows indicate HIGD2A signal in SC\nI+III\n2\n+IV\nn\n.\nAlso, in Figure 4F there seems to be less NDUFA4 in the respirasome of HIGD2A-\nKO+NDUFA4 than in HIGD2A-KO+EV, but looking at the level of NDUFA4 in monomeric\nCIV or the ATP5A loading control it does not seem like the difference would be ascribed to\nloading different amount of material: what would be the explanation for this?\nWe thank the reviewer for this insightful comment. We quantified the three blue native\nimmunoblot experiments and calculated the average NDUFA4 signal relative to WT in both\nholo-CIV and SCs I+III2+IVn. Based on this analysis, we did not detect statistically significant\ndifferences between the HIGD2A+EV and HIGD2A+A4 groups (Figure R2). The differences\ninitially observed in Figure 4F may have resulted from experimental variability, such as uneven\n8\nantibody or ECL incubation across the membrane. We have therefore replaced the NDUFA4\nblot in Figure 4F with a more representative example.\nFigure R2: NDUFA4 distribution in SC I+III +IV and CIV in HIGD2A-KO. Distribution of\n2 n\nNDUFA4 in CIV and CIV-containing supercomplexes analyzed by BN-PAGE of isolated mitochondria\nfrom WT and HIGD2A-KO cells overexpressing HIGD2A (2A), NDUFA4 (A4), or carrying an empty\nvector (EV) in three independent experiments. The NDUFA4 signal in both SC I+III +IV and holo-CIV\n2 n\nwas normalized by ATP5A signal and plotted in the bar graph relative to WT (mean ± SD of 3 independent\nexperiments).\nLines 417-423: there are no bar graphs in the figure, but indeed it would be good to see them.\nWe thank the reviewer for noticing this mistake. We have added the bar graph to Figure 4B\nshowing HIGD2A signal quantification in SCs, CIV and Sub CIV in NDUFA4-KO cells\nrelative to WT cells.\nLines 459-463: can the authors point to activity measurements proving that in absence of\nNDUFA4 oxygen reduction is less efficient?\nWe agree with the reviewer on the relevance of analyzing CIV activity in the absence of\nNDUFA4. Accordingly, we performed spectrophotometric measurements of CIV activity and\noxygen consumption analyses in NDUFA4-KO cell lines. The results of these experiments\nhave been included in the revised manuscript as Figure 3D–F. We have also added the\ncorresponding description and interpretation of these new data in the Results and Discussion\nsections. Additionally, we included a new section in the Methods to detail the protocols used\nfor these experiments.\nLine 366-372\nenzymatic activity in NDUFA4-KO cells. Consistent with previous reports, the absence of\nNDUFA4 led to an approximately 50% reduction in CIV activity, as determined by both\nspectrophotometric assays (Figure 3D) and in-gel activity staining (Figure 4H). This reduction\ncorrelated with a comparable (~50%) decrease in endogenous respiration in NDUFA4-KO cells\nrelative to WT HEK293T cells (Figure 3E, F).\nLines 464-466: how is the data helping understand the underlying pathogenic mechanisms of\nLeigh syndrome? What kind of information are the current structures adding to the state of the\nart? It appears that references 68 and 69 concern patients in which NDUFA4 is absent, or\nalmost absent, so how are the presented data helping to explain the pathogenic mechanisms\nof Leigh syndrome?\nWe appreciate the reviewer’s observations and have revised the text to clarify how our data\ncontribute to the understanding of NDUFA4 disruption-associated forms of Leigh syndrome.\nThe following paragraph has been included in the updated version of the manuscript:\nLine 509-515:\nNDUFA4 mutations have been identified in patients suffering from Leigh syndrome associated\nwith CIV deficiency. Our structural and biochemical data provide a mechanistic basis whereby\nNDUFA4 loss disrupts the final maturation of CIV, impairing its activity and mitochondrial\nrespiration without necessarily abolishing respirasome assembly. By delineating the temporal\nsequence and regulation of CIV assembly, our findings establish a direct link between\nstructural perturbations and the molecular pathogenesis of mitochondrial disorders.\nFigure S1: The particle numbers don’t add up. The consensus refinement indicates 301k\nparticles, from which the local refinements are performed. But the total of the four CIV classes\nis around 334k particles, so it is not clear where the additional particles come from.\nAdditionally, it appears that only a subset of the particles were used to refine CI (168k particles)\nand CIII (213k particles). In the Methods section, the authors state that “the focus mask further\nremoved the poor alignment particles”, but this does not seem very likely because in the\nrespirasome CI and CIII usually drive the alignment, partly because the membrane arm of CI\nand the proximal protomer of CIII form the “rigid core” of the respirasome and also because\nthe eight FeS clusters of CI and the six hemes of CIII provide strong densities that aid the\nparticle alignment. It is therefore strange that CI and CIII would be poorly aligned in the\nrespirasome, particularly CI which “loses” almost half of the particles from the consensus\nrefinement to the focused refinement. As there is no data shown for the processing steps leading\nto the final maps of CI and CIII, one cannot see how the removed classes look like, or at which\nstages they were removed. It is therefore important that the authors show exactly how the data\nwas processed for CI and CIII. Since so many particles are removed, is it possible that other\nintermediates, perhaps lacking the N-module of CI, are present in the dataset and were\ndiscarded as “poorly aligned”? Or are the discarded particles corresponding to the open state\nof CI? Also, what is the overlap between discarded particles for CI and CIII? Meaning, are the\ndiscarded particles coming from the same respirasome particles? If so, is it possible that\nthose are generally damaged particles, which therefore should not be considered for CIV refinement\neither? When focused refinements are run starting from a consensus map, to boost the\nresolution, one would expect that the number of particles does not change because this is the\npool that corresponds to the structure of interest, in this case the respirasome.\nRemoving one\n10\nthird (as was done for CIII) or half of the particles (as was done for CI) is unexpected and\nappears unjustified, therefore needs to be clarified and supported by data.\nThank you for these useful suggestions. We have now carefully reviewed data processing and\nupdated Supplementary Figure 1 with more information. The “poorly alignment detaset” is not\na correct term and has been removed. In the revised Supplementary Figure 1, we show the 4\nclasses of complex I and III after 3D classification by using the focus masks. For complex I,\nthe two classes display better structural features corresponding to a higher resolution map. Two\nclasses were then combined for CTF refinement, local refinement, followed by structural\nmodeling. After combining two classes, we now have 236,346 particles of complex I for the\nfinal map. This map has been deposited as EMDB-54784. We also update it in the data\navailability part of the manuscript.\nSame for complex III, the unused particles and classes after 3D classification display worse\nstructural features than the main class (213,731 particles). Therefore, we kept using this main\nclass for the final map and structural modeling.\nWe have modified and added following sentences in the Method part of the manuscript to\nclarify:\nLine 745-752:\nFor CI and CIII refinement, the 3D classification using the focus mask (Supplementary\nFigure 1) further discarded the subsets of particles displaying minor populations, resulting in\n236,346 particles and 213,731 particles, respectively, for the final refinement. It should be\nnoted that the unused particles exhibited lower resolution structural features or appeared to\nrepresent minor conformational states. These were not further analyzed in this study to focus\non predominant structural states as the conformational change analysis of CI and CIII have\nbeen intensively studied previously 40,42-44,85,86.\nLines 676-678: Along the same line, in the methods section it is stated that the CIV focused\nclassification was done with 6 classes, then the 6 classes should be shown in the\nsupplementary figure, also indicating which classes were pooled based on their overlapping\nfeatures.\nAfter carefully reviewing the data processing, we corrected the number of classes to eight, and\nthe maps have been shown in the updated Supplementary Figure 1.\nFigure S1 lacks:\n1) the map-to-model FSC curves for each modelled structure\n2) the half-map FSC curve for the consensus map\n3) the local resolution depictions (i.e. surface colored by local resolution), as well as\n4) the angular distribution plots for each focused map and the consensus map.\nThe description of how the plots and colored maps are obtained should then also be added to\nthe methods section. The methods section should also state whether the maps were sharpened\nand if so how.\nPoints 3 and 4 are particularly important because the provided maps don’t look like they\ncorrespond to the reported resolutions, so the local resolution coloring would clarify which\nregions of the maps really reach the reported resolution and the angular distribution would\nshed light on a potential anisotropy issue, which might contribute to explaining why the maps\ndon’t look like they correspond to the reported resolution.\nAs the “class 2” and “class 3” complex IV maps provided don’t look anisotropic, it might\n11\nalso be that the weird appearance of the ccp4 maps is a sharpening artifact, which is why it\nwould be important to declare whether the maps were sharpened.\nThank you for indicating these important points on data processing and refinement process.\nThe FSC curve for the consensus map is included in Supplementary Figure 1. We have now\nmade a new Supplementary Figure 2 with Gold standard Fourier shell correlation (GSFSC)\ncurves, the angular distribution plots, the local resolution depictions for all 6 maps that we\nprocessed. We also added map-to-model FSC curves for 4 modeled structures.\nFour maps, including maps for CI, CIII, class 1, and class 4 of CIV, were sharpened using\nphenix.auto_sharpen tool in Phenix. These sharpened maps were used for further model\nbuilding. While maps for class 2 and class 3 were kept as original maps from the local\nrefinement job.\nWe have added this information in the Method section.\nLine 710-712:\nThe final maps of CI, CIII, class 1 and class 4 of CIV from local refinement jobs were subjected\nto autosharpen job in Phenix v1.21.1, and the output maps were used for model building.\nMinor comments\nFigure S4: in panels F and G, the subunit to which Y244 and K319 belong (COX1\npresumably) should be indicated in the legend.\nInformation has been added to the figure and figure legend.\nAll figures displaying densities (Figs. 1, S2, S3, S4) should indicate the contour used in the\nlegend.\nAll figures displaying densities now contain the contour level indicated in each panel or figure\nlegend.\nLine 604: The section is titled “immunoprecipitation”, but lines 606-628 feature the description\nof sample purification for cryo-EM. I suggest this section is thus separated and titled “cryo-\nEM sample preparation”\nWe agree and as suggested by the reviewer, a new section “Cryo-EM sample preparation”\n(Line 685) has been added and separated from the previous one.\nLegend to Fig S2: state that NDP stands for NADPH, as this is not clarified.\nThis sentence is updated in the figure legend of Supplementary Figure 3 (Supplementary\nSupplementary Figure 2 in the previous version).\nElectrons are transferred from NADPH (NDP) to the flavin mononucleotide (FMN) in the\nhydrophilic domain and then sequentially down a series of Fe-S clusters (orange-yellow\nspheres).\nFig 2D: the figure, or legend, lack reference to the scalebar used to calculate the electrostatic\npotential.\nAdded in line 316-318:\nrespectively, and white color represents neutral residues.\nReviewer #2 (Remarks to the Author):\nThe assembly of the respiratory chain and supercomplex formation plays important roles in\nhuman health and disease. This paper describes a cryo-EM study of human respirasomes,\npurified using an engineered tag on a complex IV subunit. The analysis revealed I2III2IV1\nsupercomplexes with high-resolution structures of fully assembled CI and CIII, while CIV\ndensity is weak. Local refinement and classification of the flexibly attached CIV yielded\ndistinct classes, containing either the assembly factor HIGD2A and/or the subunit NDUFA4,\nwhich is the last subunit to be added to the complex.\nIn this interesting paper the authors make a convincing case that the removal of HIGD2A is\nnecessary for insertion of NDUFA4, as their positions partially overlap. However, the authors\ndo not mention what, if any, role HIGD2A plays in respirasome formation. Also, it is not\nclear if acting as a “placeholder” for NDUFA4 is the only function of HIGD2A or if it plays a\nrole in the attachment of the COX3 module, as previously suggested. The paper would be\nstrengthened if these issues were addressed.\nWe thank the reviewer for their positive and constructive comments.\nOur study builds on previous biochemical work that clearly established HIGD2A’s role in\nCOX3 insertion. This function was carefully analyzed in earlier studies9-11 , and our current\nstructural findings show close interaction between HIGD2A and COX3, which supports and is\nconsistent with this proposed role.\nWe acknowledge that structural analysis of a HIGD2A KO would further confirm its function\nin COX3 insertion. However, designing such a study poses significant technical challenges.\nLoss of HIGD2A would likely affect the early stages of complex IV assembly, potentially\nresulting in unstable or incomplete assembly intermediates—even in the context of the\nsupercomplex—making such an experiment hard to visualise structurally. While this remains\nan important avenue for future work, it is beyond the scope of the current study.\nInstead, we focused on a novel and unexpected observation: the dynamic interplay between\nHIGD2A and NDUFA4. To determine the sequence of their insertion and their\ninterdependence, we performed additional analyses using KO models, confirming that\nNDUFA4 functionally replaces HIGD2A during complex IV maturation.\nIn the revised Discussion, we have highlighted previous studies describing the relationship\nbetween HIGD2A and COX3. We also emphasize the speculation that HIGD2A may act as a\nplaceholder until the appropriate NDUFA4 isoform is incorporated. This could be particularly\nrelevant under varying physiological conditions, as both HIGD2A and NDUFA4 isoforms are\nregulated by oxygen availability.\nRegarding the functional relevance of our results to respirasome formation, we are happy to\nreport that our data support the notion that complex IV (CIV) assembly occurs within the\ncontext of the whole respirasome. However, we currently lack direct evidence on how\ndisruption of HIGD2A or NDUFA4 affects the assembly of the other components of the\nrespirasome. We therefore consider this an important avenue for future investigation.\nSpecific comments:\n13\n1. The protein was purified with a tag on CIV, so presumably yielding both respirasomes and\nsingle CIV. However, the latter is never mentioned. Are the particles visible in the\nmicrographs? If so, why are these not analysed? If not, how can this be explained?\nWe thank the reviewer for this important point. During data processing, we specifically\nsearched for particles corresponding to Complex IV alone. However, we did not obtain any 2D\nclass averages that resembled isolated CIV. Initially, we attempted to use material from FLAG\nco-immunoprecipitation directly for grid preparation and collected a small dataset, but the\nresulting sample was highly heterogeneous, and subsequent 2D classification failed to yield\nuseful classes. To improve sample quality, we optimized the preparation workflow. In\nparticular, we included additional post-immunoprecipitation centrifugation steps, including a\n16-hour spin at 55,000 rpm in a TLA55 rotor, which enriched for higher-molecular-weight\nassemblies while depleting smaller complexes. We believe that this step substantially reduced\nthe presence of isolated CIV, thereby explaining their absence in our cryo-EM data. This choice\nwas made to minimize heterogeneity and maximize the recovery of well-resolved respirasome\nparticles for high-resolution structural analysis. Details of these steps are provided in the\nMethods section under \"Sample preparation for cryo-EM”.\n2. The introduction, p. 4 states “it has been proposed that HIGD2A promotes the formation of\nrespirasome assembly intermediates by coordinating the association of CIV assembly modules,\nrevealing divergent assembly pathways for individual and supercomplexed CIV”. Is there any\nevidence for this? For a distinction it would be necessary to study individual CIV as well. It is\nnowhere shown how CIV is oriented in the respirasome and what subunits interact with CI or\nCIII.\nRecently, there has been increasing evidence that the formation of the respirasome happens\nthrough a cooperative assembly mechanism. Regarding the binding of CIV precursors to SCs,\nbiochemical analyses in CIV-deficient cell lines showed that SC CICIII2 is stabilized through\nits interaction via assembly factor HIGD2A with an atypical MT-CO3 module bound to MT-\nCO1 10,15,16.\nWe agree with the reviewer that it is necessary to study individual CIV and how HIGD2A\naffects its assembly. However, we were unable to visualize CIV-like particles, despite many\nefforts in particle picking and 2D classification. As mentioned in the previous comment, the\nsample of COX14-FLAG coimmunoprecipitation has been investigated biochemically,\nshowing interactions with individual and complexed CIV; however, our cryoEM failed to\nidentify CIV like particles.\nWe added Supplementary Video 1 to show the flexibility of CIV relative to CI and CIII\ncomplex as analyzed by 3D variability. On the other hand, the orientation of CIV relative to CI\nand CIII had been well studied in the previous reports of the mammalian respirasomes 17-20.\n3. Further to that, the model in Fig. 5 does not show what induces CIV attachment to the\nrespirasome.\nWe thank the reviewer for this insightful question. Two models of respirasome assembly have\nbeen proposed: (1) the plasticity model, in which individual complexes assemble first and then\nassociate into supercomplexes; and (2) the cooperative model, where supercomplexes form\nthrough the association of incomplete intermediates 16,21. The coexistence of free MRC\n14\ncomplexes and respirasome is demonstrated by both reported high-resolution structures and\nblue native-PAGE (BN-PAGE). Recently, there has been a growing body of evidence\nsuggesting binding of CI, CIII and CIV precursors before the maturation of the individual\n2\ncomplexes, which corresponds to the second model 15,22,23. Capturing such intermediate states\nat high resolution remains technically challenging due to their low abundance and transient\nnature, although biochemical approaches such as BN-PAGE provide indirect support.\nRegarding the binding of CIV precursors to SCs, recent data, particularly from CIV-deficient\ncell lines, suggest that an atypical COX3 module, stabilized by HIGD2A, can associate with\nCI–CIII₂, forming a potential precursor (“CI–CIII₂ plus”) prior to full CIV maturation 10,15.\nWhile our assembly model in Fig. 5 is inspired by this hypothesis, we refrain from specifying\nthe precise sequence of COX module incorporation, as our data do not directly capture\nintermediate states. To avoid overinterpretation, we grouped the three COX modules together\nand clearly stated in the legend that their assembly order remains unresolved.\n4. p. 12: The story about the flexibility at the K-pathway entrance is confusing. The region is\nsaid to typically have high B factors, indicating flexibility, nevertheless distinct conformations\nare assigned to these uncertain densities. Particularly suspicious is Glu62: a negatively charged\nside chain typically has no cryo-EM density (unless the charge is compensated by a nearby\npositive charge like a salt bridge or ion ligand) and the side chain can't be modeled correctly\neven in a rigid structure. The absence of the glutamate sidechain is obvious in Suppl. Fig. 4F\nand G (although just showing density for one residue does not give an indication of the level\nof flexibility in this region). Thus, the suggestion that the conformation of Glu62 in a bovine\nCIV structure in GDN represents an intermediate state between the two human structures is a\nvery weak argument (Fig. 3C). If the authors feel that there is a real conformational change in\nthis region, better figures will be needed showing the actual densities, not just models.\nOtherwise, this section needs to be toned down. Further to suppl. fig. 4F,G:\nplease indicate\nwhich subunits the highlighted residues belong to. Glu62 is misspelled as Glu61 in the legend.\nWe thank the reviewer for raising this point. Both reviewers expressed concern about this\nsection, and we agree that our original description may have been an overinterpretation.\nOf note, we have updated Supplementary Figure 5 (previously Figure S4) to include panels\nS5H and S5I, which present the B-factors of the COX2 protein in both HIGD2A- and\nNDUFA4-bound complex IV. The entrance of the K-pathway, particularly Glu62, displayed a\nhigher B factor in the HIGD2A-bound state compared to the NDUFA4-bound state. However,\nas this evidence is not sufficiently strong and we don’t provide functional relevance, we have\nmoderated our discussion and removed the sentence from the results:’ Additionally, it suggests\nthat NDUFA4 binding may regulate proton transfer through the K-pathway.’\nLine 501-508\nOur structures suggest that NDUFA4 binding may influence proton transfer by stabilizing the\nentry point of the K-pathway. In this scenario, oxygen reduction at the CIV catalytic center\nwould proceed efficiently only in the fully mature holoenzyme, linking NDUFA4 binding to\nfull functional activation of CIV, both as an individual complex and within the respirasome.\nFurther investigation will be required to define the mechanistic link between NDUFA4 or\nHIGD2A incorporation, K-pathway stabilization, and efficient oxygen reduction in isolated\nCIV and in the respirasome.\n5. Figure 1B: please indicate how CIV is oriented relative to the respirasome shown in 1A.\nnow updated the figure legend.\n6. Cryo-EM methods (p.23):\na. What is the protein concentration?\nWe added this information to ‘Sample preparation for cryoEM’ part in the Methods.\nLine 708-709\nGrids were prepared using protein at a concentration of 1 mg/ml.\nb. What microscope was used?\nWe have now added this information to ‘Cryo-EM data acquisition and data processing’ in the\nMethods.\nLine 718-721\nAll cryo-EM data collection (Supplementary Table 1) was performed with EPU (Thermo\nFisher Scientific) operated on Titan Krios G3i transmission electron microscope (Thermo\nFisher Scientific) at 300 kV in the Karolinska Institutet’s 3D-EM facility.\nc. Please add “total electron dose” of 35 e-/Å2.\nThank you! Added.\nd. How many particles were picked?\nWe added this information to ‘Cryo-EM data acquisition and data processing’ part in the\nMethods.\nLine 731:\nA total of 2,508,182 particles were initially picked from two data sets.\ne. “bad alignment particles” and “poor alignment particles”: please rephrase.\nWe have updated this information in ‘Cryo-EM data acquisition and data processing’ part in\nthe Methods.\nLine 738-742:\nAfter homogeneous refinement of these particles, 3D classification without mask followed by\niterative heterogeneous refinements was performed to get rid of classes displaying broken\ndensities, resulting in 336,847 particles yielding 3.1 Å resolution after homogeneous\nrefinement (Supplementary Figure 1).\nLine 744-752:\nMasked refinements of the respective SC intermediates resulted in map resolutions of 2.9 Å\nfor CI, 2.8 Å for CIII and 3.1 Å for CIV. For CI and CIII refinement, the 3D classification\nusing the focus mask (Supplementary Figure 1) further removed the subsets of particles\ndisplaying minor populations, resulting in 236,346 particles and 213,731 particles,\nrespectively, for the final refinement. It should be noted that the unused particles exhibited\nlower resolution structural features or appeared to represent minor conformational states. These\nwere not further analyzed in this study to focus on predominant structural states as the\nconformational change analysis of CI and CIII have been intensively studied previously 40,42-\n44,81,82.\nf. What is a “local refinement volume”?\nAnswer:\nWe are sorry for causing confusion. The focus masks are described in ‘Cryo-EM data\nacquisition and data processing’ part in the Methods and Supplementary Figure 1.\nLine 745-748:\nFor CI and CIII refinement, the 3D classification using the focus mask (Supplementary figure\n1) further removed the subsets of particles displaying minor populations, resulting in 236,346\nparticles and 213,731 particles, respectively, for the final refinement.\ng. Global or local CTF refinement?\nWe performed both global and local CTF refinement. We have now updated the Method\nsection with this information.\n7. Model building (p. 24):\na. Densities for lipids were manually fitted using references: please explain.\nWe carefully inspected the lipid molecules in the previously reported models of complex I, III\nand IV [PDB: 8OM1, 5XTD for CI; 5XTE, 8UGD for CIII; 5Z62, 8UGL for CIV] and used\nthem as references to fit the lipids into our maps. We now add reference for each PDB structure.\nb. “The ligands, cofactors and modifications were manually inspected as suggested in Uniprot\nand experimentally determined structures reported previously”. Unclear.\nDuring refinement of each protein or subunit as components of CI, CIII and CIV, we carefully\ninspected each protein one by one by using the sequence and binding cofactors, which have\nbeen listed in the database in Uniprot. On the other hand, several previously reported models\nof complex I, III and IV have been inspected as reference models.\nWe now rephased this sentence for clarification:\nLine 780-783:\nThe ligands, cofactors and modifications of proteins or subunits in individual complexes were\nmanually inspected according to Uniprot database and experimentally determined structures\nreported previously (PDB: 8OM1 for CI; 8UGD for CIII; and 5Z62 for CIV).\nc. HIGD2A was manually built with an AlphaFold structure as reference. Please remove\n“manually”.\nRemoved.\n8. Suppl. fig. 1: The workflow needs more details/images: exemplary micrographs, 2D class\naverages, number of particles picked initially, initial models.\nWe now updated Supplementary Figure 1 and added new Supplementary Figure 2, which\ntogether contain more details about data processing and refinement.\nReferences:\ncomplex I. Science 370, eabc4209, doi:doi:10.1126/science.abc4209 (2020).\ncomplex I and conformational transitions accompanying Q-site occupancy. Nat.\nCommun. 13, 2758, doi:10.1038/s41467-022-30506-1 (2022).\nmammalian mitochondrial complex I. Nat. Struct. Mol. Biol. 29, 172-182,\ndoi:10.1038/s41594-022-00722-w (2022).\nmechanism: are the open states of mammalian complex I identified by cryoEM resting\nstates or catalytic intermediates? Curr. Opin. Struct. Biol. 77, 102447,\ndoi:https://doi.org/10.1016/j.sbi.2022.102447 (2022).\nmechanism of function. Biochim. Biophys. Acta 1827, 1278-1294,\ndoi:https://doi.org/10.1016/j.bbabio.2012.11.008 (2013).\nbc1 and b6f Complexes of Energy-Transducing Membranes. Chem. Rev. 121, 2020-\n2108, doi:10.1021/acs.chemrev.0c00712 (2021).\nNuclear-Encoded Protein. Cell 167, 471-483.e410, doi:10.1016/j.cell.2016.09.003\n(2016).\nSynthesis with Cytochrome c Oxidase Assembly, Cause Fatal Neonatal Lactic\nAcidosis. The American Journal of Human Genetics 90, 142-151,\ndoi:10.1016/j.ajhg.2011.11.027 (2012).\nHuman Mitochondrial Complex IV. Mol. Cell. Proteomics 19, 1145-1160,\ndoi:10.1074/mcp.RA120.002076 (2020).\nRoles of Mitochondrial HIGD1A and HIGD2A in Respiratory Complex and\nSupercomplex Biogenesis. Cell Reports 31, doi:10.1016/j.celrep.2020.107607 (2020).\nRegulation of Cytochrome c Oxidase Biogenesis and Function. Cells 9, 2620 (2020).\nN-terminal Homolog Rcf3 in <em>Saccharomyces cerevisiae</em>*<sup>\n</sup>. J. Biol. Chem. 291, 23769-23778, doi:10.1074/jbc.M116.734665\n(2016).\nfunction of S. pombe complex IV with bound respiratory supercomplex factor.\nCommunications Chemistry 6, 32, doi:10.1038/s42004-023-00827-3 (2023).\nin Bovine Heart Mitochondria and the Composition of Respiratory Chain\ndoi:https://doi.org/10.1074/jbc.M106474200 (2001).\ncomplex IV and stabilization of respiratory supercomplexes. EMBO J. 39, e103912,\ndoi:https://doi.org/10.15252/embj.2019103912 (2020).\nrespiratory chain. Trends in Biochemical Sciences 47, 999-1008,\ndoi:https://doi.org/10.1016/j.tibs.2022.07.005 (2022).\ndoi:10.1038/nature19359 (2016).\nsupercomplexes. Nature 537, 644-648, doi:10.1038/nature19774 (2016).\nSupercomplex I1III2IV1. Cell 167, 1598-1609.e1510, doi:10.1016/j.cell.2016.11.012\n(2016).\nRespiratory Megacomplex I2III2IV2. Cell 170, 1247-1257.e1212,\ndoi:10.1016/j.cell.2017.07.050 (2017).\nmitochondrial respiratory chain. Nature Reviews Molecular Cell Biology 23, 141-161,\ndoi:10.1038/s41580-021-00415-0 (2022).\nmediated maturation of human mitochondrial complexes I and IV. EMBO J. 39,\ne102817, doi:https://doi.org/10.15252/embj.2019102817 (2020).\nrespirasome formation. Cell Reports 35, doi:10.1016/j.celrep.2021.108963 (2021).\n19"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I thank the authors for thoroughly considering and addressing my comments. I am satisfied with the revised version of the\nmanuscript and recommend publication without further revision.", "reviewer_2": "The authors have properly addressed most of my comments. One issue remains: the interpretation of differences in the K\npathway between complex IV maps in the presence of NDUFA4 or HIGD2A. Line 360: “The density for Glu62 of COX2 in the\nHIGD2A-bound CIV suggests uncertainty regarding side chain conformation, consistent with high flexibility (Supplementary\nFigure 5F). Interestingly, this region appears slightly more ordered in the presence of NDUFA4 (Supplementary Figure\n5G), indicating a potential stabilizing effect.”. In the revised manuscript the authors have added local resolution plots, which\nclearly show that the resolution in this region is poor in both maps. But in fact, looking at the maps provided by the authors,\nboth CIV maps also suffer from anisotropic resolution, which is not visible from a static picture. Due to all these factors, even\nthe backbones can’t be reliably interpreted in either map, let alone sidechains. And, as stated in my original review,\nnegatively charged sidechains typically have no cryo-EM density. So the “uncertainty regarding side chain conformation” is\nnot even due to the low resolution.\nFurther, the authors state that “this region appears slightly more ordered in the presence of NDUFA4 (Supplementary Figure\n5G), indicating a potential stabilizing effect”. Sfig. 5 F and G show density for Glu62 only, in 5G at a very low contour level\nwhere there is a blob of density assigned to the sidechain. The figure is missing all the context of the maps, where there is\nno indication of “better ordering” in one of them.\nFigure 3C shows the “conformational change” of Glu62, including a previously published structure (8D4T from EMD-27196).\nI checked that map and although it has better resolution than the ones from the present study, there is no density for the\n(charged) Glu62 sidechain and its conformation in the structure is based on guesswork.\nIn short, the authors do not provide any evidence for a conformational change or lower flexibility due to NDUFA4 binding.\nThe region is simply flexible in all structures. It would be best if the whole story (line 340-364) is eliminated, including Figure\n3C and Sfig. 5F and 5G."}, "author_response": "Response to reviewers’ comments\nReviewer #1 (Remarks to the Author):\nI thank the authors for thoroughly considering and addressing my comments. I am\nsatisfied with the revised version of the manuscript and recommend publication\nwithout further revision.\nWe thank the reviewer for their evaluation and are pleased that our revisions have\naddressed their comments.\nReviewer #2 (Remarks to the Author):\nThe authors have properly addressed most of my comments. One issue remains: the\ninterpretation of differences in the K pathway between complex IV maps in the\npresence of NDUFA4 or HIGD2A. Line 360: “The density for Glu62 of COX2 in the\nHIGD2A-bound CIV suggests uncertainty regarding side chain conformation,\nconsistent with high flexibility (Supplementary Figure 5F). Interestingly, this region\nappears slightly more ordered in the presence of NDUFA4 (Supplementary Figure\n5G), indicating a potential stabilizing effect.”. In the revised manuscript the authors\nhave added local resolution plots, which clearly show that the resolution in this\nregion is poor in both maps. But in fact, looking at the maps provided by the authors,\nboth CIV maps also suffer from anisotropic resolution, which is not visible from a\nstatic picture. Due to all these factors, even the backbones can’t be reliably\ninterpreted in either map, let alone sidechains. And, as stated in my original review,\nnegatively charged sidechains typically have no cryo-EM density. So the “uncertainty\nregarding side chain conformation” is not even due to the low resolution.\nFurther, the authors state that “this region appears slightly more ordered in the\npresence of NDUFA4 (Supplementary Figure 5G), indicating a potential stabilizing\neffect”. Sfig. 5 F and G show density for Glu62 only, in 5G at a very low contour level\nwhere there is a blob of density assigned to the sidechain. The figure is missing all\nthe context of the maps, where there is no indication of “better ordering” in one of\nthem.\nFigure 3C shows the “conformational change” of Glu62, including a previously\npublished structure (8D4T from EMD-27196). I checked that map and although it has\nbetter resolution than the ones from the present study, there is no density for the\n(charged) Glu62 sidechain and its conformation in the structure is based on\nguesswork.\nIn short, the authors do not provide any evidence for a conformational change or\nlower flexibility due to NDUFA4 binding. The region is simply flexible in all structures.\nIt would be best if the whole story (line 340-364) is eliminated, including Figure 3C\nand Sfig. 5F and 5G.\nWe agree with the reviewer that this was the most speculative part of the manuscript,\nand we have removed it from the final version.\nFigure 3C, Supplementary Figures 5F and 5G, and the corresponding text (lines 340–\n364) have been deleted. We also removed Supplementary Figures 5H and 5I, which\npresented subtle differences in B-factor values for the COX2 protein in the HIGD2A-\nbound and NDUFA4-bound complex IV.\nAdditionally, we revised the corresponding section of the Discussion (previously lines\n501–508) as follows:\nPrevious text:\nThe incorporation of NDUFA4 is synchronized with the release of HIGD2A from CIV,\ncompleting the assembly of both isolated and supercomplexed CIV. Our structures\nsuggest the possibility that NDUFA4 binding may influence proton transfer by\nstabilizing the entry point of the K-pathway. In this scenario, oxygen reduction at the\nCIV catalytic center would proceed efficiently only in the fully mature holoenzyme,\nlinking NDUFA4 binding to full functional activation of CIV, both as an individual\ncomplex and within the respirasome. Further investigation will be required to define\nthe mechanistic link between NDUFA4 or HIGD2A incorporation, K-pathway\nstabilization, and efficient oxygen reduction in isolated CIV and the respirasome.\nRevised text:\nThe incorporation of NDUFA4 is synchronized with the release of HIGD2A from CIV,\ncompleting the assembly of both isolated and supercomplexed CIV. It is tempting to\nspeculate that NDUFA4 binding may influence proton transfer. In this scenario, oxygen\nreduction at the CIV catalytic center would proceed efficiently only in the fully mature\nholoenzyme, linking NDUFA4 binding to full functional activation of CIV, both as an\nindividual complex and within the respirasome. Further investigation will be required\nto define the mechanistic link between NDUFA4 or HIGD2A incorporation and efficient\noxygen reduction in isolated CIV and the respirasome."}]

Decision letter