**1. Introduction**

Complex I (CI, NADH:ubiquinone oxidoreductase) is the first and largest enzyme of the mitochondrial respiratory chain in humans. It catalyzes the transfer of electrons from NADH to coenzyme Q10, which is coupled to the translocation of protons from the mitochondrial matrix into the intermembrane space. Recently, the structures of the entire CI in *Yarrowia lipolytica*, *Ovis aries*, and *Bos taurus* were reported at a resolution of 3.6 to 4.2 Å, describing in detail the central subunits that execute this bioenergetic function [1–3]. Mammalian CI consists of 45 subunits, seven of which are encoded by the genes localized in mitochondrial DNA [4–6]. Therefore, CI deficiency can originate from both mitochondrial or nuclear DNA mutations, which leads to its heterogeneous features [7,8].

Since the discovery of pathogenic mitochondrial DNA (mtDNA) point mutations [9,10] and deletions [11] in the year 1988, more than 309 gene defects have been reported to date, and this number continues to grow [12]. Mitochondrial diseases can be grouped into (i) disorders of oxidative phosphorylation (OXPHOS) subunits and their assembly factors; (ii) defects of mitochondrial DNA, RNA, and protein synthesis; (iii) defects in the substrate-generating upstream reactions of OXPHOS; (iv) defects in relevant cofactors; and (v) defects in mitochondrial homeostasis [13]. Mitochondrial diseases occur at an estimated prevalence of 1 in 5000 live births, and are collectively the most common inborn error of metabolism [14,15]. CI deficiency is the most frequent mitochondrial disorder among inborn errors of metabolism, and is characterized by clinical and genetic heterogeneity [16] including Leber's hereditary optic neuropathy (LHON) [10], mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes (MELAS) [17], and Leigh syndrome (LS) [18]. In addition, the level of heteroplasmy of mtDNA mutations can vary and is dynamic between cells in the same organism or tissue, and the proportion of mutant mtDNA molecules determines both the penetrance and severity of expression of disease [19]. Recently, Idebenone was designated as the first orphan drug to treat LHON by the European Medicines Agency (EMA product number: EMEA/H/C/3834). Idebenone functions as a mitochondrial electron carrier and bypasses CI to directly transfer electrons to mitochondrial complex III (CIII) [20].

In this study, we applied an integrative proteome and metabolome profiling approach to investigate the molecular and cellular consequences of pathogenic mutations in two core subunits of mitochondrial CI. The first nuclear gene, *NDUFS1*, encodes the NADH-ubiquinone oxidoreductase 75 kDa subunit, the largest subunit of CI that accommodates three iron–sulfur clusters in the N-module, which binds and oxidizes NADH [21,22]. The second gene, *MT-ND5*, is located in the mtDNA and encodes NADH-ubiquinone oxidoreductase chain 5, which represents one of the core subunits in the P-module, wherein the proton translocation takes place. It is located at the distal end of the CI transmembrane arm and facilitates proton translocation [23,24].

The first patient was a girl, who carried a mutation in the mitochondrial gene *MT-ND5* (m.12706T>C). This missense mutation caused a single amino acid substitution of p.Phe124Leu. The second patient was a boy. He carried point mutations in *NDUFS1* (c.683T>C and 755A>G, compound heterozygous), which caused amino acid substitutions of p.Val228Ala and p.Asp252Gly. Identical mutations in both genes have been previously reported to cause a pathogenic phenotype [25–30]; however, the molecular and cellular consequences of these mutations were largely unknown. Here, we explored and compared the proteome and metabolome profiles of patients and control skin fibroblasts to elucidate (i) if the global and OXPHOS-specific protein and metabolite abundances were altered, (ii) if the assembly of CI and the formation of the mitochondrial respirasome was influenced, (iii) if enzymatic activities of OXPHOS were regulated, (iv) if reactive oxygen species (ROS) production was changed in these distinct CI mutations versus una ffected controls, and (v) whether the electron tunneling rate in NDUFS1 was impaired because of the mutation between iron–sulfur clusters N4 and N5.

#### **2. Materials and Methods**

## *2.1. Ethics Statement*

The study protocol conformed to the guidelines of the Declaration of Helsinki. Studies with primary human cell lines were approved by the local ethics committee "Ethikkommission Land Salzburg" and written informed consent was provided by the patients' guardians for skin biopsies.

#### *2.2. Mutations in Patients*

The first patient carried a missense mutation in the mitochondrial DNA (gene *MT-ND5*, m.12706T>C), which lead to an amino acid substitution (p.Phe124Leu) in the ND5 subunit of complex I. Sanger sequencing revealed a 70% mutation load in cultivated skin fibroblasts. This mutation has

been reported in patients with Leigh syndrome [25–27]. The second patient carried two distinct point mutations (c.[683T>C];[755A>G]) in the nuclear gene *NDUFS1* (NM\_005006.7), which caused amino acid substitutions (p.[Val228Ala];[Asp252Gly]) in the NDUFS1 subunit of complex I. This patient was compound heterozygous for these two missense mutations.
