*3.4. Proteome Profiling*

Fibroblasts harboring the *MT-ND5* and *NDUFS1* mutations were compared to two individual healthy controls in triplicates by label-free quantification (LFQ) in a total of 60 LC-MS/MS runs. We identified more than 5363 protein groups with at least two peptides per protein group (Table S2). We then compared both mutations individually to controls and filtered for 100% valid values in at least one group and replaced missing values from the normal distribution. This resulted in a total of 4030 protein groups for the *MT-ND5* patient and 3893 for the *NDUFS1* patient versus controls (Table S3).

The reproducibility of the biological replicates was tested by Pearson correlation and visualized in a multi-scatter plot for all experiments. The Pearson correlation coe fficients were highly similar, ranging from 0.959 to 0.994 in controls, 0.989 to 0.992 in the *MT-ND5* mutation, and 0.988 to 0.994 in the *NDUFS1* biallelic mutations (Figure S3), indicating very robust replicates.

Statistical analysis by an unpaired two-sample *t*-test identified 1535 significant proteins and 1090 significantly regulated proteins after Benjamini–Hochberg (BH) correction for multiple testing (*p*-value ≤ 0.05, FDR ≤ 0.05) in the *MT-ND5* mutation versus controls, as visualized in a volcano plot (Figure 3A). In the case of the *NDUFS1* mutations, 324 significantly deregulated proteins were found after the *t*-test, and 145 were identified upon BH correction (*p*-value ≤ 0.05, FDR ≤ 0.05) (Figure 3B).

**Figure 3.** *Cont*.

**Figure 3.** Abundance ratios of CI subunits mapped onto the CryoEM structure of human CI, PDB: 5XTD. (**A**) Protein abundance ratios between mutND5 and controls. Mitochondrial proteins according to Human MitoCarta2.0 are shown in orange, OXPHOS proteins in blue, and CI subunits in red. The dotted line indicated the threshold of significance (FDR ≤0.05) in the two-sample *t*-test after Benjamini–Hochberg correction. Protein names are presented for significantly regulated CI subunits. (**B**) Protein abundance ratios between mutNDUFS1 and controls. Same legend as in (**A**). (**C**) The mutation in *MT-ND5* did not lead to a general change of the abundance of CI subunits, but a specific loss of the N-module was identified in the *NDUFS1* patient. The inset indicates the relative position of the N-module, NDUFS1, and ND5 in CI. n.d., no data.

#### *3.5. Gene Set Enrichment Analyses Reveal Glycolysis is Upregulated in the MT-ND5 Mutation and the Respiratory Chain is Down-Regulated in the NDUFS1 Mutations*

We applied the pathway enrichment tool GSEA to assess whether a priori defined sets of proteins showed statistically significant, concordant differences between *MT-ND5* and *NDUFS1* versus controls, respectively. Pathways with significant *p*-values (≤0.05) and FDR q-values (≤0.05) are listed in Tables S4 and S5. In the patient with the *MT-ND5* mutation, pathways comprising proteins of the cytoskeleton, the extracellular matrix, cytosolic tRNA aminoacylation, and glycolysis and gluconeogenesis were significantly upregulated (Table S4).

Many structural and cytoskeleton proteins were enriched in the *MT-ND5* patient, such as myosins MYL9 and MYLK, actin ACTA2, tropomyosins, TPM1, CALD1, MYL6, TPM4, FN1, and collagens, reflected in the respective pathways of muscle contraction, focal adhesion, and collagen formation.

The pathway "cell cycle" was significantly down-regulated in cells harboring the *MT-ND5* mutation, including six of the proteins of the mini-chromosome maintenance complex (MCM) responsible for DNA replication, (threefold, see Table S4).

In the case of the patient with *NDUFS1* mutations, the pathway including cytoskeleton components was only significantly increased at a nominal *p*-value (Table S5).

The respiratory electron chain was the only significantly down-regulated pathway in the patient carrying *NDUFS1* mutations (Table S5). To further shed light on the substructures of CI, we performed a pathway analysis with manually created gene lists for all modules of CI. This analysis identified a significant and specific decrease only in the N-module (*p* ≤ 0.001, q-value ≤0.05, Table S5), including the subunits NDUFA7 (threefold), NDUFV2 (fourfold), NDUFS1 (sixfold), and NDUFV1 (tenfold) (Figure 3B).

To visualize this dysregulation, all protein abundance ratios of CI subunits between patients and controls were mapped in a three dimensional structure of CI (PDB: 5XTD). The inlet indicated the position of subunit ND5 and NDUFS1 in CI (Figure 3C). Indeed, the N-module in the *NDUFS1* patient was the only region to be severely reduced, whereas the *MT-ND5* patient showed no changes (Figure 3C). This indicated that the stability of the entire N-module was a ffected, most likely because of fast degradation of misfolded or not integrated subunits of CI.

#### *3.6. The Rate of Electron Tunneling between the Iron–Sulfur Clusters N4 and N5 of NDUFS1 Was Predicted to Decrease Dramatically in a V228A Mutant*

We examined the electron transfer between the iron–sulfur clusters N4 and N5 in subunit NDUFS1 using the method of tunneling current theory, as was previously described for a bacterial enzyme [37]. It revealed that the residue Val228 was critical for bridging the electron transfer between the N4 and N5 clusters, as electrons tunnelled primarily through this relatively bulky residue. Our simulations for an ovine enzyme showed that the mutation p.Val205Ala, with a smaller alanine substitution, had a dramatic e ffect on the rate of electron transfer by reducing it by 35-fold (Figure 4). It is interesting that the e ffect of mutation of this critical valine residue was predicted to occur earlier in *Thermus thermophilus* [37]. These changes were predicted to occur in the remaining small fraction of fully assembled CI.

**Figure 4.** Electron tunneling pathway of the electron transfer reaction between iron–sulfur clusters N4 →N5 of the wild-type and *in silico* mutated CI in *Ovis aries*, PDB 5LNK. Solid blue arrows indicate through-space jumps in the primary electron tunneling pathways. Through-space distances in Ångstrom are shown next to the arrows. Relative color density indicates the contribution of the corresponding atom/bond in electron transfer reaction. The mutation resulted in a reduced rate of electron transfer *kWT ET* /*kV*205*<sup>A</sup> ET* = 35 for the ovine enzyme.

#### *3.7. Decreased Stability of CI in Mutated NDUFS1 Prevents the Formation of Supercomplexes*

To elucidate the consequences of the specific loss of the N-module in the mutated *NDUFS1* cell line for the formation of supercomplexes, we solubilized OXPHOS proteins under mild conditions using digitonin, and performed blue native PAGE followed by western blot. Only a small fraction of supercomplexes were formed compared to controls (Figure 5A–D). In addition, a major part of CIII was not incorporated into supercomplexes, which was the stoichiometric assembly of CI, III, and IV (Figure 5D). In contrast, no assembly errors were identified in the *MT-ND5* patient. Succinate dehydrogenase (complex II, CII) was used as a loading control and showed no differences between samples (Figure 5E).

**Figure 5.** Formation of respiratory chain supercomplexes and in-gel activity assay of CI. Blue native PAGE and western blot detection of respiratory chain enzymes solubilized with digitonin. (**A**) Antibody against NDUFS1, a core subunit of the N-module in CI; (**B**) antibody against NDUFS2, a core subunit of the Q-module in CI; (**C**) antibody against NDUFB8, an accessary subunit of the P-module in CI; (**D**) antibody against UQCRC2, a core subunit of CIII; (**E**) antibody against SDHB, a subunit of complex II (CII), which serves as a loading control. (**F**) In-gel activity assay (IGA) of CI. Ctrl: control; MHM: mouse heart mitochondria, as molecular weight marker and positive control; SC: supercomplexes; III, complex III (CIII); IV, complex IV (CIV).

To test the functionality of the partly assembled respirasome, an in-gel activity assay of CI was performed and revealed that there was almost no enzymatic activity in the patient with *NDUFS1* mutations (Figure 5F).

#### *3.8. Isolated CI Deficiency in Both Patients*

Enzymatic measurements of the respiratory chain enzymes and citrate synthase (CS) were performed in the homogenates of muscle biopsies for both patients [40,54]. These were normalized to CS and were compared to reference values (Table 1) [31]. In both cases, the CI enzyme activity was below the reference values, while all other complexes showed values within the range of the references, indicating an isolated CI deficiency.

#### *3.9. Live Cell Respiration Assays Revealed a Low Oxygen Consumption Rate in Both Patients*

The respiration rate measurements in live cells (Figure 6A) elucidated that the basal respiration, ATP-linked respiration, maximal respiration, and the spare respiration capacity were less than 50% compared with controls (Figure 6C–F). In contrast, the basal glycolysis rate was significantly higher (*p*-value ≤ 0.05) in patients than in the controls (Figure 6G). The glycoPER (Figure 6B), which is the PER (proton efflux rate) contributed by glycolysis, was significantly higher (*p*-value ≤ 0.05) in the patients (Figure 6H,I). Thus, a clear metabolic shift from aerobic respiration to glycolysis was observed in both mutated cell lines (Figure 6J), which is in concordance with our proteomics data for the patient with the *MT-ND5* mutation, where the glycolytic pathway was upregulated.

**Figure 6.** Cellular respiration assays showing mitochondrial dysfunction and increased glycolytic activities in both patients. (**A**) Oxygen consumption rate profile. (**B**) Proton efflux rate (PER) profile. (**C**) Basal respiration rate at the beginning of the assay. (**D**) ATP-linked respiration before the addition of the inhibitors. (**E**) Maximal respiration after carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) was added. (**F**) Calculated spare respiratory capacity. (**G**) Basal glycolysis rate at the beginning of the assay. (**H**) Total PER before the addition of the inhibitors. (**I**) Percentage of PER from glycolysis. (**J**) The ratio between mitochondrial oxygen consumption rate (OCR) and glycolytic PER as an indicator of the cellular energetic profile. Error bars: (**A**) and (**B**): mean ± SD; (**C**–**J**): mean ± 95% confidence intervals. One-way ANOVA: *p*-value < 0.01 (\*\*); *p*-value < 0.001 (\*\*\*); ns, not significant.
