2.2.2. NDUFS6

The gene for NDUFS6 is located in chromosome 5 and contains four exons. The protein has 124 amino acids before the N-terminal 28 are cleaved upon entry to the mitochondrion. The original mass is 13.7 kDa. The protein is bound to the matrix arm and has two domains connected by a short alpha-helix, residues 67–75 (see Figure 5). The N-terminal domain, residues 29–66, is closer to the membrane and primarily contacts subunits NDUFA9 and NDUFA12. The C-terminal domain, residues 76–124, contains several short beta-strands and is in contact with core subunits NDUFS1, NDUFV2, NDUFS2, and NDUFS8. A zinc ion is bound by three cysteines and one histidine that are part of a C–X8–H–X7–C–X2–C motif with Cys87, Cys112, Cys115, and His96. The significance of the bound zinc is not clear, but this subunit is found not only in the fungal *Y. lipolytica* enzyme [29,30] but also in some bacterial enzymes (e.g., *Paracoccus denitrificans*) [31]. In cell culture, the knockout of NDUFS6 results in a mild assembly defect in which complexes of somewhat smaller size with a

reduced oxidative phosphorylation capacity are seen [11]. In *Y. lipolytica*, the knockout of the NDUFS6 gene results in a complex lacking NDUFA12 and the N4 FeS cluster. Complementation with constructs lacking one of the zinc binding residues failed to regenerate the wild-type activity, suggesting a role in the incorporation of this FeS cluster [29].

The first mutations associated with NDUFS6 were described in 2004 [32]. Three individuals presented with lethal neonatal mitochondrial complex I deficiency and died within days of birth. Two were siblings with homozygous mutations, c.186 + 2 T > A, which affected splicing, leading to 26 bp of intronic RNA incorporated at the exon 2/3 junction, causing a frameshift and a protein predicted to be 71 amino acids. A small amount (3%) of normally spliced transcript was also found. The third individual was also homozygous, with a deletion of 4.75 kb that removed exons 3 and 4. This individual died five days after birth.

A mutation in one of the residues that bind zinc, p.Cys115Tyr, was found in two different families [33]. The affected individuals, two from each family, presented with fatal neonatal lactic acidemia and died within a few days of birth. All were homozygous for this allele.

Two additional mutations in NDUFS6 were identified, first in 2017 [34] and then again in 2019 [35] when a more extensive analysis was done. Both were characterized as having Leigh syndrome, and the individuals were compound heterozygous, carrying both mutant alleles, c.309 + 5 G > A and c.343 C > A (p.Cys115Arg). The individual analyzed in 2019 lived for eleven months. It was determined that the splicing error led to a transcript missing exon 3, but that a small amount of normally spliced form was present. Complex I assembly was very limited, as indicated by BN gel electrophoresis. The mutation of Cys115 to Arg or Tyr could disrupt the folding of the protein and its binding to the matrix arm, which could have led to the loss of the FeS cluster N4, as was shown to happen in the fungal *Y. lipolytica* enzyme [29].

#### 2.2.3. NDUFA9

The gene for NDUFA9 is located on chromosome 12. It is encoded as a protein of 377 amino acids, about 42.5 kDa, but the N-terminal 35 amino acids are cleaved upon entry to the mitochondrion. The protein contains two domains, one being a Rossmann fold with an NADP<sup>+</sup> bound to the C-terminal ends of the parallel beta-strands of the central sheet. A second domain, which contacts the NADP+, is largely alpha-helical. The second domain is largely C-terminal, and the Rossmann fold is largely N-terminal, except for two helices have been domain-swapped (see Figure 5). This protein is related to a family of short chain dehydrogenases, as first identified by Fearnley and Walker [36]. It is also one of the proteins that undergoes conformational changes in the active/deactive transition, as seen in mouse (PDB id = 6g2j, 6g72) [37] and sheep open and closed structures [38]. NDUFA9 is found at the junction of the Q-module and the membrane arm. It primarily has contact with core subunits NDUFS3 and NDUFS7, as well as with NDUFS4, NDUFS6, and NDUFA6. It also has minor contact with the core subunits ND1, ND3, ND6, NDUFS1, and NDUFS8. There is no evidence that this subunit exhibits any enzyme activity.

A human cell line was established with a knockout of NDUFA9 using transcription activator-like effector nucleases (TALEN) technology [39]. A clear assembly defect was discovered in which an 880 kDa complex was transiently seen, but a more stable 600 kDa complex was later found. The latter complex had the membrane arm subunits and some of the Q-module subunits of the matrix arm. In a more comprehensive study of all supernumerary subunits [11], the same group later established that levels of N-module subunits decreased the most in cell lines with the NDUFA9 knockout. The offered interpretation was that NDUFA9 is important for stabilizing the binding of the N-module to the Q-module during assembly. The complex seen in cells with the NDUFA9 knockout was smaller than those seen when NDUFS4 or NDUFS6 were knocked out, indicating that the Q-module was also destabilized.

Two missense mutations have been discovered in patients. The first was p.Arg321Pro, a homozygous mutation found in a boy of consanguineous parents [40]. He had complications in respiration after birth, with lactic acidosis and vision and hearing loss. He died after one month of respiratory failure, and it was classified as Leigh syndrome. An analysis of cultured fibroblasts showed very low levels of NDUFA9, and several other subunits, as well as essentially no assembled complex I in BN gel electrophoresis. Arg321 is found in the NADP<sup>+</sup> binding pocket near the nicotinamide end but not in contact with it. It does not contact any other subunits, as it is completely buried. Therefore, it is likely that the substitution to Pro would disrupt the folding of the subunit and render it unable to properly bind to the assembling complex I.

‐

The second discovered mutation also involved a homozygous Arg mutation, p.Arg360Cys [41]. This patient first exhibited symptoms of dystonia at age seven, leading to a loss of speech and becoming wheelchair-bound. His conditions stabilized in adulthood. A biochemical analysis did not reveal any metabolic disease, but an MRI of his brain revealed atrophy consistent with Leigh syndrome. An analysis of fibroblast mitochondria revealed reduced levels of complexes I and IV by BN gel electrophoresis, as well as reduced levels of several complex I subunits by immunoblotting. Complex I activity was 17–61% of control samples. Complementation with wild-type NDUFA9 confirmed that this mutation was causative for the reduced complex I activity. Arg360 is near the C-terminus of the subunit and is sandwiched between Trp361 of NDUFA9 and Tyr78 of ND6, a core subunit. A similar arrangement is seen in the mouse structure of the active conformation [37], except that Glu80 of ND6 is also involved. This junction between NDUFA9 and ND6 appears to be disrupted in the deactive conformation, as shown in the mouse structure [37] in which the C-terminus of NDUFA9 is not visible and is perhaps disordered. A similar situation has been seen in the higher resolution structures from sheep [38] in which the loops between NDUFA9 and ND6 are formed in the closed structures (PDB id = 6zko and 6zkc), while these loops, including Arg360 are disordered in the open structures (PDB id = 6zkd, 6zke, 6zkf, 6zkp, and 6zkr). Therefore, it is possible that even if the p.Arg360Cys subunit can assemble, it might be less able to undergo conformational changes during turnover, and so activity would decrease. ‐ ‐ ‐ ‐

#### *2.3. ND1-Module Subunits ‐*

Three membrane subunits from the "heel" of complex I are described next: NDUFA1, NDUFA3, and NDUFA13, as shown in Figure 6. These subunits are part of the ND1-module for assembly. Each crosses the membrane once. NDUFA3 interacts with the transmembrane domain of NDUFA13, while NDUFA1 interacts with NDUFA13 in the IMS. ‐

‐ **Figure 6.** Location of NDUFA1, NDUFA3, and NDUFA13. Most of Complex I is colored gray. Core subunits in the ND1-modules are colored light blue (ND1). NDUFA1, NDUFA3, and NDUFA13 are shown in ribbons, with NDUFA1 colored blue, NDUFA3 colored orange, and NDUFA13 colored pink. The two views are rotated 180◦ relative to each other. The structure is from PDB id = 5xtd [9].
