**4. NDUFS4-Linked Complex I Dysfunction at the Molecular Level**

Several lines of evidence indicate that NDUFS4 plays a role in the late stage of complex I assembly [66,68,113,114]. In animal models and patient cell lines, quantitative mass spectrometry showed that deletion of NDUFS4 caused an increase of assembly factor ND-UFAF2 and induced a near complete loss of accessory complex I subunit NDUFA12 [115]. In BN PAGE, an 830 kDa subcomplex harbouring NDUFAF2, but lacking the N module, has been observed. However, in intact tissue, substantial rotenone sensitive Q reductase

activity was found, which argues against the complete loss of the N module under in vivo conditions [116]. It is interesting to note that integration of complex I into supercomplexes appears to have a stabilizing function for complex I lacking NDUFS4 [117].

We have studied the impact of a NDUFS4 gene deletion on complex I function and assembly in the aerobic yeast *Y. lipolytica* [118]. We found that in the yeast KO strain, complex I levels were decreased and ubiquinone (Q) reductase activity in membranes was reduced. Complexome profiling of intact mitochondria showed that assembly factor NDUFAF2 was bound to complex I, but in clear contrast to the situation observed for mammalian species [115], we did not find a substantial decrease of NDUFA12. In the yeast system, large scale purification of complex I is straightforward. We found that in purified complex I from the NDUFS4 deletion strain, all subunits except NDUFS4 were present and the amount to NDUFAF2 was clearly substoichometric. This suggests that NDUFAF2 was only loosely attached to complex I before solubilization and was easily removed during protein purification. The purified complex showed reduced ubiquinone reductase activity while the formation of ROS under turnover conditions was increased. The EPR spectrum of mutant complex I showed a marked change in the N1b and N3 signals. The cryo-EM structure of the mutant (Figure 2) [19] offered a straightforward explanation for the biochemical and spectroscopic data. We found that the absence of NDUFS4 exposes clusters N1b and N3 to solvent. Thus, the change in EPR spectra is caused by the loss of the shielding function of the accessory subunit. Interestingly, in *T. thermophilus* complex I, the NDUFS1 subunit has an extra loop that partially matches the position of the NDUFS4 subunit in mitochondrial complex I [118]. The increased ROS formation of the mutant might be linked with the greater solvent accessibility of FeS clusters or a longer dwell-time of electrons on FMN, which is known to be critical for the generation of superoxide [119].

**Figure 2.** Structure of complex I lacking NDUFS4 and of an assembly intermediate harboring assembly factor NDUFAF2. (**A**) Cryo-EM structure of *Y. lipolytica* complex I purified from *ndufs4*∆ strain (PDB ID: 6rfs); the red arrow indicates direction of view for (**B**) and (**C**). (**B**) Detail view on NDUFS4 in wild type *Y. lipolytica* complex I (PDB ID: 6rfr), direction of view (see (**A**)). (**C**) Same as (**B**) for *ndufs4*∆ mutant (PDB ID: 6rfs); FeS clusters N1b and N3 are solvent exposed. (**D**) Structure of complex I assembly intermediate purified from *Y. lipolytica ndufs6*∆ strain (PDB ID: 6rfq). The assembly intermediate harbors assembly factor NDUFAF2 and all subunits, except NDUFS6 and NDUFA12. For clarity, only NDUFAF2 and NDUFS4 are shown in color. The position of NDUFAF2 matches the position of NDUFA12 in wild type complex I.

#### **5. The Role of NDUFS4, NDUFS6, and NDUFA12 in Complex I Assembly**

The intricate assembly pathway of mammalian complex I has been studied in detail [120–122]. Five submodules are initially formed and then combined in a stepwise process to yield complete complex I. At least 15 assembly factors are known to associate with submodules and play an indispensable role in the assembly process [120]. Assembly factor NDUFAF2 was originally identified as a c-Myc controlled mitochondrial protein (Mimitin) with similarity to complex I subunit NDUFA12 [123]. Whole genome subtraction of fermentative and non-fermentative yeasts gave strong indications that NDUFAF2 is a complex I assembly factor and a null mutation of the associated gene was shown to cause progressive encephalopathy [110]. Analysis of mutants in the fungus *Neurospora crassa* [114] and complementation assays using human mitochondria derived from patients [113] showed that NDUFAF2 function is tightly associated with the attachment of the N module and that NDUFS4, NDUFS6, and NDUFA12 must work together to release the assembly factor in the final step of complex I biogenesis. Since NDUFAF2 and NDUFA12 are paralogs, it had been proposed that both polypeptides occupy the same position in the mature enzyme complex and in the preceding assembly intermediate [64]. We have shown that deletion of the gene encoding NDUFS6 in *Y. lipolytica* caused accumulation of an assembly intermediate that lacked NDUFA12, while NDUFAF2 remained firmly bound [63]. The Q reductase activity of the NDUFS6 KO mutant was reduced to 44%. Mutations in the zinc binding site stalled complex I assembly to varying degrees. Pathogenic mutations in NDUFS6 have been reported (Table 2) and exchange of a cysteine ligand of the metal binding site was shown to cause fatal neonatal lactic acidosis [103]. Taking advantage of straightforward His-tag affinity purification of *Y. lipolytica* complex I, we obtained a preparation of the assembly intermediate of sufficient quality for high-resolution structure determination by cryo-EM (Figure 2) [19]. The structure shows that in the assembly intermediate NDUFAF2 in fact matches the position of NDUFA12 in mature complex I. The NDUFAF2 structure also clashes with the position of NDUFS6. The structure thus offers a straightforward explanation for why NDUFS6 and NDUFA12 are required for the release of NDUFAF2. At first sight, the role of NDUFS4 was less clear, because the subunit appeared to be separated from the assembly factor binding site. Interestingly, no cryo-EM density was observed for a sequence stretch of about 100 amino acids in the C-terminal part of the assembly factor indicating disorder. A finger-like protrusion of NDUFS4 penetrates a narrow cleft between the N and Q modules and comes close to the site where the NDUFAF2 structure is unresolved. We have proposed that in the assembly intermediate NDUFS4 has already pushed out a domain of the assembly factor which becomes flexible after detachment from the complex. The major part of the assembly factor remains bound because NDUFS6 is lacking and the NDUFS6/NDUFA12 tandem cannot be formed for complete removal of NDUFAF2. In the NDUFS4 KO, a weak association of complex I with NDUFAF2 is possible because the protein surface occupied by this accessory subunit in the wild type is still available for the assembly factor in the mutant. The C-terminal end of the assembly factor binds to NDUFS1 and anchors the N module. We propose that before the binding of NDUFS4, the un-modelled sequence stretch of the assembly factor is bound to the assembly intermediate and forms a platform for the docking of the N module. Thus, the C-terminal part of the assembly factor guides the N module to its attachment site, while the N terminal domain is responsible for a stable connection with the nascent complex. These results give a consistent picture for *Y. lipolytica*, but cannot explain why NDUFAF2 remains firmly bound in mammalian NDUFS4 KO cells [115]. We propose that there is no fundamental difference in the N module assembly but that only the relative contribution of NDUFS4, NDUFS6, and NDUFA12 for the detachment of NDUFAF2 is different. In *Y. lipolytica*, NDUFS4 plays a minor part, while, in mammals, the lack of NDUFS4 precludes NDUFAF2 detachment, which in turn blocks the association with NDUFA12. This may also explain a weaker binding of the N module in the mutant complex I.

A recent report showed that the N module is turned over faster than the rest of complex I [124]. The N module is thought to be more exposed to oxidative damage because of superoxide formation at the FMN cofactor [119,125]. Selective exchange of dysfunctional N module is advantageous, because it has a lower energetic cost than *de novo* synthesis of the complete enzyme complex. Interestingly, the three subunits discussed here are among the group of subunits with the highest exchange rate in agreement with their role in the attachment of the N module.
