**1. Introduction**

Mitochondrial complex I (proton pumping NADH:ubiquinone oxidoreductase) is the largest and most intricate membrane protein complex of the respiratory chain [1–4]. It is a redox-driven proton pump that couples electron transfer from NADH to ubiquinone (Q) with vectorial proton translocation across the inner mitochondrial membrane. With a proton pump stoichiometry of 4 H<sup>+</sup> per NADH consumed, complex I contributes about 40% of the proton motive force that drives ATP synthase. Mitochondrial complex I from a broad range of species can reversibly switch from an active A form into an inactive D form [5,6]. The A/D transition is thought to protect against excessive formation of reactive oxygen species [7,8]. The structure of complex I has been determined by X-ray crystallography [9,10] and cryo-EM [1,11–16] and is now well described.

Mammalian complex I comprises 45 subunits [17]. We have established *Yarrowia lipolytica* as a yeast genetic model organism to study eukaryotic complex I [18]. *Y. lipolytica* complex I comprises 43 subunits of which 40 are orthologues of mammalian complex I [19]. In this review, we use the nomenclature for human complex I also for orthologous proteins from other organisms (Table 1). The large number of polypeptides is divided into central subunits and accessory subunits [20,21]. The 14 central subunits are conserved from bacteria to man and are assigned to three functional modules [21]. The N module (central subunits NDUFS1, NDUFV1, NDUFV2) for NADH oxidation and the Q module (central subunits NDUFS2, NDUFS3, NDUFS7, NDUFS8) for Q reduction are located in the matrix

**Citation:** Kahlhöfer, F.; Gansen, M.; Zickermann, V. Accessory Subunits of the Matrix Arm of Mitochondrial Complex I with a Focus on Subunit NDUFS4 and Its Role in Complex I Function and Assembly. *Life* **2021**, *11*, 455. https://doi.org/10.3390/ life11050455

Academic Editors: Giorgio Lenaz and Salvatore Nesci

Received: 11 April 2021 Accepted: 14 May 2021 Published: 19 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

arm of complex I (Figure 1, Table 1). In the membrane arm, the P module (central subunits ND1 to ND6 and ND4L) for proton translocation is subdivided in a proximal P<sup>P</sup> and a distal P<sup>D</sup> module [22]. The genes for the seven central subunits of the membrane arm represent a substantial part of the mitochondrial genome. All other complex I subunits and all assembly factors are encoded by nuclear DNA. The N module harbors the NADH oxidation site with the initial electron acceptor FMN. The N and Q modules together comprise eight FeS clusters [23,24]. Cluster N1a is thought to have a function for transient storage of electrons to prevent excessive ROS formation and/or to control NADH binding in the active site [25]. The other seven FeS clusters are arranged in an electron transfer chain connecting the NADH oxidation site with the Q reduction site [26]. Cluster N2 is the immediate electron donor for Q. In contrast to other Q reactive enzymes, the Q reduction site of complex I is buried in the protein structure and is located remotely from the membrane phase [27]. The hydrophobic Q has to transit through a tunnel into the Q module to receive electrons from N2 [9,28,29]. It is generally accepted that the energy driving the proton pumps is released in the Q module. However, the coupling mechanism of complex I has remained controversial.

The large majority of accessory subunits is only found in eukaryotic complex I. A notable exception are subunits NDUFS4, NDUFS6, and NDUFA12 that are already present in complex I from α-proteobacteria [30]. The accessory subunits are arranged around the core of central subunits [10,16,31,32]. In general, their function is less clear, but in many cases, severe complex I assembly defects were found after knock out (KO) of individual genes coding for accessory subunits in human cell lines [33].

Mutations causing a broad spectrum of pathological conditions were reported for central and accessory subunits and assembly factors, and they have been reviewed recently [32,34–36].

Here, we focus on accessory subunit NDUFS4 in the matrix arm of complex I and the interplay of accessory subunits with assembly factor NDUFAF2 during the attachment of the N module.


**Table 1.** Subunits of the peripheral arm of respiratory complex I.

α

**Figure 1.** Functional modules of complex I and accessory subunits of the matrix arm. (**A**) The central subunits of complex I are assigned to functional modules for NADH oxidation (N module, orange), ubiquinone reduction (Q module, green), and proton pumping (P module, cyan). (**B**) Accessory subunits of the matrix arm of human complex I (PDB ID: 5xtd) are shown in color, all other subunits are shown in gray. (**C**) Accessory subunits of the matrix arm of *Y. lipolytica* complex I (PDB ID: 6rfr; color code as in (**B**)); the sulfur transferase subunit ST1 is not part of the model; note that FV3 is not present in *Y. lipolytica* complex I.

#### **2. Accessory Subunits of the Matrix Arm in Yeast and Mammalian Complex I**

The matrix arm of mammalian and yeast complex I comprises 10 accessory subunits. Overall, the same set of subunits is found, but subunit NDUFV3 of mammalian complex I is not present in the yeast enzyme complex. NDUFV3 is the only subunit for which tissue specific isoforms have been reported [37–39]. On the other hand, only *Y. lipolytica* complex I is associated with the sulfur transferase subunit ST1 [40,41]. Binding of ST1 is substoichiometric and the deletion of the ST1 gene has no impact on complex I function or biogenesis.

NDUFA9 is the largest accessory subunit of the matrix arm. It has the fold of a short chain dehydrogenase [42] and binds NADPH [43]. The cofactor is present in all structures of the eukaryotic complex with sufficient resolution and is therefore a tightly bound component of the subunit [1,11]. The NADPH molecule is too far away from the nearest FeS cluster to allow electron transfer and its function remains unknown. It has been shown recently that NDUFA9 binds the head groups of several phospholipid molecules, which is remarkable for a subunit of the peripheral arm [19]. The subunit is thought to undergo a conformational change in the A/D transition [44] and the relaxation of the protein structure in the C-terminal domain of the subunit has been reported for the D form of mammalian complex I [45].

Mammalian complex I binds two copies of the mitochondrial acyl carrier protein (ACPM) subunit NDUFAB1. In contrast, the yeast enzyme comprises two different but closely related ACPM variants [46]. In all cases, a fatty acid is appended to the phosphopantethein group of the ACPM [47,48]. This fatty acid is inserted into the interior of a mitochondrial LYR (Lys-Tyr-Arg motif) protein that forms a heterodimer with an ACPM [32,49,50]. The mitochondrial LYR proteins were initially implicated in FeS cluster biogenesis [51], but are now recognized to be associated with different macromolecular complexes in the mitochondrion [49]. ACPM/LYRM heterodimers are bound to the Q module of complex I (NDUFAB1α/NDUFA6) [50] and to the tip of the membrane arm (NDUFABβ/NDUFB9) [52]. It is interesting to note that free NDUFAB1 has an essential

function in mitochondrial fatty acid synthesis to generate the octanoic acid precursor for lipoic acid [53]. The ACPMs associated with complex I carry longer chain fatty acids and a regulatory function is debated [54]. We have shown that binding of the LYRM protein NDUFA6 to the matrix arm is essential for the Q reductase activity [50]. More recently, we determined the structures of NDUFA6 mutants and showed that single exchanges at the contact site with the functionally important ND3 loop have a strong impact on the interface region of the matrix and membrane arms [55].

NDUFA5 has been noticed in connection with the A/D transition, because the interface of this subunit and accessory subunit NDUFA10 must rearrange during deactivation [45]. Since NDUFA10 is lacking in complex I from *Y. lipolytica*, the longer lifetime of the A form in mammalian complex I might be connected with this specific structural feature [13].

NDUFA2 has a thioredoxin-like fold, but its function has remained unclear. In mammals, the subunit has two cysteine residues, but in *Y. lipoytica*, only one cysteine is conserved.

The three subunits NDUFS4, NDUFS6, and NDUFA12 are distinguished by the fact that they are already found in complex I from α proteobacteria [30]. NDUFS4 has attracted a lot of attention, because it is a hot spot for pathogenic mutations. Knock-out mouse models (*Ndufs4* KO) are widely used to study Leigh syndrome (LS) [56,57]. Moreover, NDUFS4 can be singled out because, in mammalian species, it harbors a canonical serine phosphorylation site [58]. However, analysis of bovine complex I by mass spectrometry did not provide evidence for phosphorylation of the subunit [59,60]. Phosphorylation is thought to play an important role during import and/or maturation of the precursor protein [61,62]. NDUFS6 has a zinc binding site [63]. It is interesting to note that NDUFA12 is a paralog of assembly factor NDUFAF2 [64]. Several lines of evidence have indicated that the interplay of subunits NDUFS4, NDUFS6, and NDUFA12 with assembly factor NDUFAF2 is critical for the attachment of the N module to nascent complex I.

#### **3. Leigh Syndrome and the** *Ndufs4* **KO Mouse Model**

In humans, inactivation of the NDUFS4 gene on chromosome 5 is known to cause severe neurologic disorders [65–68]. In most cases, LS or Leigh-like syndrome is diagnosed (Table 2) [69–75]. LS is a rare disease with a prevalence of roughly 1:40.000 live births and a generally poor prognosis [76–78]. A recent meta-analysis showed that 35% of LS cases are associated with defects in respiratory complex I [79]. In 2016, a ratio of 22 cases of NDUFS4 linked LS for a group of 198 patients with complex I-linked LS was reported [73]. Genotyping of microsatellite DNA markers and array-comparative genomic hybridization has been utilized for diagnosis and might be used for patients with a high pre-test probability in the future [80,81]. Blue native (BN) PAGE consistently revealed abnormal assembly profiles in skin fibroblasts from affected patients and was proposed as a reliable and specific screening method [82]. *Ndufs4* KO mouse models as well as human and murine cell lines have been used extensively to study LS and to explore strategies to counteract the pathophysiological consequences of complex I deficiency [56,57]. Attempts to alleviate disease progression such as expression of plant NDH-2 [83], administration of redox-modulators [84], or targeting of NAD<sup>+</sup> -metabolism [85,86] have been reported. Inhibition of mTOR by rapamycin was shown to dramatically improve survival and health in *Ndufs4* KO mice [87], probably by rescuing a dysfunctional α-ketoglutarate/glutamate/glutamine metabolic axis [88]. The metabolite α-ketoglutarate is thought to sustain sufficient OXPHOS capacity and substrate level phosphorylation even when complex I activity is compromised [89]. In addition, there is evidence that glutamatergic neurons, in particular, drive disease development [88]. The link between mTOR inhibition and the neuron-specific neurotransmitter metabolism opens up a further possible explanation for the positive effect of rapamycin. mTOR is present in two distinct complexes, mTORC1 and mTORC2. mTORC2 was initially described as rapamycin insensitive; however, chronic rapamycin treatment is thought to decrease the formation of new functional mTORC2 [90], resulting in a decrease of PKC-β-dependent pro-inflammatory signaling [91]. Rapamycin treatment thus exerts its positive effect via the inhibition of both mTORC complexes, resulting in changes in metabolism and a decreased

tendency to inflammation. In another promising approach at the preclinical stage, it was shown that hypoxia treatment with 11% O<sup>2</sup> not only ameliorated symptoms but, in fact, led to the reversal of neurological impairment in the *Ndufs4* KO mouse model [92–94]. It was recently demonstrated that hypoxic breathing normalizes a detrimental hyperoxia in brain tissue, while activation of the hypoxia-inducible factor (HIF) is not a crucial factor [95]. A new perspective on LS has recently been opened by the observation that switching from glycolytic metabolism to OXPHOS is critical for early neuronal morphogenesis [96]. Defective metabolic reprogramming due to mutations in OXPHOS complexes is thought to be incompatible with normal brain development and might lead to early termination of pregnancy in more cases than previously known.


**Table 2.** Summary of pathogenic mutations in NDUFS4, NDUFS6, NDUFA12, and NDUFAF2.

Gene therapy approaches in the *Ndufs4* KO mouse model were also pursued as an alternative to pharmacological therapy options [111,112]. Adeno-associated viral vector (AAV)-based gene replacement showed promising results in *Ndufs4* KO mice, but differences in the blood brain barrier between mouse and human are still an obstacle for future clinical applications [112].
