**3. Results**

#### *3.1. Substituted Amino Acids in ND5 and NDUFS1 are Highly Conserved from Bacteria to Human*

A protein sequence alignment across multiple species showed a highly conserved phenylalanine at position 124 in the subunit ND5, which was substituted to leucine in one patient (Figure S1A). The heteroplasmy level of this mutation was 70% in cultivated skin fibroblasts, which is in agreemen<sup>t</sup> with other reports, displaying a very severe phenotype [25–27]. Furthermore, Phe124 is localized in the fourth transmembrane helix of subunit ND5, which is close to the proposed proton translocation channel [27] and thus may influence its structure and catalytic function. In the other patient, two highly conserved amino acids, valine at position 228 and aspartate at position 252, were substituted to alanine and glycine in NDUFS1, respectively (Figure S1B). Val228 is located between the two iron–sulfur clusters N4 and N5 in subunit NDUFS1 (protein data bank, PDB: 5XTD, human CI).

#### *3.2. Metabolome Profiling Revealed a Decrease of the GSH*/*GSSG Ratio in Both Patients*

To quantify relative di fferences in metabolite changes and to elucidate key metabolic alterations caused by mutating the *MT-ND5* and *NDUFS1* genes, we applied a targeted liquid chromatography-mass spectrometry (LC-MS/MS) approach based on multiple reaction monitoring (MRM) [33]. In total, 121 metabolites were quantified relatively (Table S1). The Pearson correlation coe fficients were highly similar, ranging from 0.968 to 0.996 in the controls, 0.984 to 0.996 in the *MT-ND5* mutation, and 0.985 to 0.991 in the *NDUFS1* mutations (Figure S2), suggesting a very good quality of the metabolite data sets. Statistical analysis by an unpaired two-sample *t*-test identified significantly regulated metabolites, and six of them were significant after Benjamini–Hochberg (BH, FDR ≤ 0.05) correction for multiple testing (*p*-value ≤ 0.05) in the *MT-ND5* mutation versus controls (Figure 1A). In the case of the *NDUFS1* mutations, 11 significant metabolites were found after the *t*-test and six were identified upon BH correction (*p*-value ≤ 0.05, FDR ≤ 0.05) (Figure 1B). Interestingly, the same metabolites were found to be significantly regulated in both patients.

Glutathione (GSH) was the metabolite with the highest decrease in both patients (12-fold in ND5, 16-fold in NDUFS1, Figure 1A,B). In contrast, oxidized glutathione (GSSG) levels were increased in both patients. The ratio between reduced and oxidized glutathione (GSH/GSSG ratio) can be used as a marker for the redox status of a cell [47–49]. The concentration ratios of GSH/GSSG between patients and controls decreased significantly (more than 35-fold) for both patients' fibroblast cells (Figure 2), thus indicating a higher level of oxidative stress. Furthermore, the polyamine N-acetylputerescine was significantly increased in both patients, also indicative for higher stress levels [50,51].

**Figure 1.** Significantly regulated metabolites between mutated *MT-ND5* and *NDUFS1* versus controls, respectively. (**A**) Abundance ratios of metabolites between mutated *MT-ND5* and controls. (**B**) Abundance ratios of metabolites between mutated *NDUFS1* and controls. Metabolites above the solid lines were considered significant after the *t*-test. Metabolites above the dashed horizontal line were significant after Benjamini–Hochberg correction (false discovery rate (FDR) ≤0.05) for multiple testing. Log2 fold changes were plotted against the -Log10 (*p*-value). Blue: metabolites involved in cellular oxidative stress response; orange: metabolites of the TCA cycle; green: CI-related metabolites.

**Figure 2.** Reduced and oxidized glutathione (GSH/GSSG) ratios of controls, ND5, and NDUFS1 mutant fibroblast cells. The log2 concentration ratios (GSH/GSSG) were compared between patients and controls. One-way ANOVA of log2 ratios was performed (*p*-value < 0.001, labeled as \*\*\*). Error bars: mean ± SD.

#### *3.3. The TCA Cycle Metabolites—Fumaric and Malic Acid—Significantly Increased in Both Patients*

Malate and fumarate are two TCA cycle intermediates that were significantly upregulated in both patients. Similar findings have been reported in patients' urine samples [52]. Lactic acid, which is converted from pyruvate, was elevated twofold in both patients. This is in concordance with frequently observed lactic acidosis in patient blood [53]. The ability of CI to oxidize NADH in both patients

seems to be limited and, as a consequence, the level of NAD+ was found to be significantly reduced (Figure 1A,B).

Furthermore, flavin mononucleotide (FMN) and riboflavin (vitamin B2, a precursor of FMN) were both decreased threefold in patient fibroblasts carrying the *MT-ND5* mutation. FMN is a prosthetic group of mitochondrial CI and accepts electrons from NADH.

The metabolome survey identified that the same metabolites were significantly regulated in both patients. These metabolites, such as GSH, GSSG, NAD<sup>+</sup>, NADP<sup>+</sup>, FMN, malate, and fumarate, are all directly or indirectly involved in, or dependent on the functionality of CI.
