**5. Conclusions**

We have characterized the molecular consequences of two distinct CI mutations that result in the stalling of electron flow within CI by two di fferent mechanisms. In the *NDUFS1* patient, destabilization of the N-module and, in addition, an interruption of electron tunneling between the iron–sulfur clusters N4–N5 of the remaining assembled CI was observed. In the *MT-ND5* patient, a dysfunctional proton channel might be less e fficient to translocate protons utilizing the energy provided by the electron transfer. The interruption of the electron flow led to electron leakage and, in turn, to increased ROS generation, as seen by the reduced GSH/GSSG ratios in both cases. Furthermore, the isolated CI deficiency induced a metabolic switch towards a glycolytic phenotype, and the imbalance of the NADH/NAD<sup>+</sup> ratio caused an identical feedback on the regulation of TCA cycle metabolites in both mutations.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/8/10/1149/s1, Figure S1: Protein sequence alignment of ND5 and NDUFS1. Mutated amino acids in patients were indicated with red triangles. Secondary structure elements are shown above the alignments. ( **A**) Sequence alignments for ND5. (**B**) Sequence alignments for NDUFS1. Figure S2: Pearson correlation of MRM-targeted metabolome data among patients and controls. Correlation coe fficients are shown in the squares. Ctrl, control; rep, replicate. Figure S3: Pearson correlation of label-free quantification (LFQ) proteome data among patients and controls. Correlation coe fficients are shown in the squares. Ctrl, control; rep, replicate. Table S1: Identified metabolite ratios between patients and controls. Fold changes for each metabolite and Benjamini–Hochberg (BH) corrected two-sample *t*-test values are indicated. Table S2: MaxQuant output file featuring the proteome profiles of fibroblasts harboring the *MT-ND5* and *NDUFS1* mutations, as well as healthy controls with LFQ intensities. Table S3: Proteome analysis by Perseus with fold changes between mutations versus controls and two-sample *t*-test significances. Table S4: Gene set enrichment analysis (GSEA) report of all upregulated (sheet 1) and downregulated (sheet 2) pathways in the patient carrying the *MT-ND5* mutation. The significant threshold was set to *p*-value ≤ 0.05, FDR ≤0.05, and is highlighted in green. Table S5: GSEA report of all upregulated (sheet 1) and downregulated (sheet 2) pathways in the patient carrying the *NDUFS1* mutations. The significant threshold was set to *p*-value ≤ 0.05, FDR ≤0.05, and is highlighted in green. Table S6: Mass spectrometry transition settings for metabolites and MRM ion ratios. RT of 0 min indicates that the metabolite was measured continuously because of the long elution time of the compound.

**Author Contributions:** Y.N. performed the experiments, analyzed the data, and prepared the figures. D.M. conceived the study. Y.N. and D.M. wrote the paper. M.A.H. and A.A.S. modeled the electron tunneling. J.A.M. provided enzymatic analyses of muscle biopsies. V.K. performed organic acid analysis and took care of a patient.

**Funding:** This research was supported by the NIH gran<sup>t</sup> GM054052 to A.A.S.

**Acknowledgments:** We appreciate David Stroud (University of Melbourne, Australia) for providing the Python script to prepare Figure 3. We thank Rainer Glauben (Charité - Universitätsmedizin Berlin) for kindly sharing the Seahorse XFe96 Analyzer. We also wish to thank the referees for a number of insightful suggestions. Our work is supported by the Max Planck Society. The work in UC Davis (A.A.S. and M.A.H.) was supported by the NIH gran<sup>t</sup> GM054052.

**Conflicts of Interest:** The authors declare no conflict of interest.
