*2.5. Subunits of the Twin C–X9–C Family in the IMS*

Complex I has four proteins that are members of the twin C–X9–C family and three with identified mutations that are described next: NDUFS5, NDUFA8, and NDUFB10, as shown in Figure 10. All are found in the IMS. They are substrates of the IMS oxidoreductase CHCHD4, also known as hMia40, which is essential for the export of FeS clusters from the mitochondrial matrix [61].

**Figure 10.** Location of NDUFS5, NDUFA8, and NDUFB10. Most of Complex I is colored gray. NDUFS5, NDUFA8, and NDUFB10 are shown as ribbons, with NDUFS5 colored red, NDUFA8 colored green, and NDUFB10 colored blue. They are found facing the intermembrane space. The two views are rotated 180◦ relative to each other. The structure is from PDB id = 5xtd [9].

#### 2.5.1. NDUFS5

‐ ‐ ‐ ‐ The gene for NDUFS5 is found on chromosome 1. The protein contains 106 amino acids, with a mass of 12.5 kDa. In cultured human cell lines, the knockout of NDUFS5 completely eliminates the assembly of complex I [11]. Though this protein was initially identified with the iron–sulfur protein fraction of bovine complex I, it is now known to be localized to the IMS and does not contact any proteins with FeS clusters. NDUFS5 is bound to the surface of the membrane and lacks transmembrane helices. It has a limited tertiary structure and includes a helix–coil–helix with two C–X9–C sequence motifs that form two disulfides between the two alpha-helices at residue pairs 33–66 and 43–56. Another alpha-helix is formed by residues 70–86, but the rest of the protein has little regular secondary structure. The N-terminal domain (2–30) primarily contacts core subunits ND2 and ND4L, and supernumerary subunit NDUFB5, while the C-terminal domain (70–106) primarily contacts core subunit ND6, and NDUFB13.

‐ ‐ ‐ ‐ ‐ One missense mutation in NDUFS5 has been reported in a compound heterozygous individual, p.Pro96Ser [22]. This individual has a second mutation in core subunit NDUFS8, p.Arg2Cys, of unknown consequence. These mutations were identified in a cohort of 103 patients with complex I deficiency. Pro96 in NDUFS5 is highly conserved among vertebrates and appears as the third Pro in an unusual sequence motif of Pro-Pro-Pro-His-His near the C-terminus. In human complex I (Video S1), Pro96 contacts Thr129 of NDUFA13. There is a nearby ion pair between Lys101 of NDUFS5 and Glu89 of NDUFA13. Somewhat different interactions can be seen in the mouse complex I in both active and deactive conformations [37]. Therefore, it seems likely that this missense mutation would disrupt local structure, but it is not clear that it would be deleterious to complex I assembly. The mutation in NDUFS8, p. Arg2Cys, should also be considered, as it might be defective, for example, in translation initiation.

#### 2.5.2. NDUFA8

The gene for NDUFA8 is located on chromosome 9. The protein has 172 amino acids with a mass of about 20 kDa, and the initial methionine is cleaved. NDUFA8 is found in the IMS and has large contact surfaces with supernumerary subunits NDUFA13, NDUFA3, and NDUFA1, and it is near core subunit ND1. It has two pairs of alpha helices, each having two pairs of Cys residues that likely form disulfides (see Figure 11). They have characteristic spacings of either 9 or 11 amino acids between the cysteine residues. In the human structure (PDB id = 5xtd), not all disulfides are shown as formed, but it is likely that they do form in the IMS: C36–C66, C46–C56, C78–C110, and C88–C100. In the mouse structures (PDB id = 6g2j and 6g72), they are shown as disulfides [37]. The N-terminal residues 2–22 are extended and interact with NDUFA13, NDUFS5, and NDUFA1. The C-terminal region extends from residues 113 to 172 without a regular secondary structure. It makes an interesting junction with NDUFC2 and NDUFB5 in a conserved multi-centered ion pair including Arg166 of NDUFA8, Asp86 of NDUFC2, and Glu153 of NDUFB5. ‐

‐ ‐

‐ ‐ ‐ **Figure 11.** Structural features of NDUFA8, a member of the twin C–X9–C. The proteins are portrayed in ribbons. NDUFA8 is colored green, NDUFC2 is colored gold, and NDUFB5 is colored blue. In NDUFA8, the eight Cys residues that form disulfide bonds are shown in space-filling and colored yellow. The sites of three mutations, Arg47Cys, Glu109Lys, and Arg135Gln, are shown in space-filling and colored black. NDUFB5 makes contact near Arg47, and both NDUFB5 and NDUFA8 contact NDUFC2. The site of one mutation in NDUFC2, His58Leu, is shown in space-filling and colored red.

‐ At least three missense mutations have been described in NDUFA8. The p.Arg47Cys mutation was described in a patient that was homozygous, having received a mutant allele from each parent, who were heterozygous and asymptomatic [62]. This individual presented with developmental delay and epilepsy, and they were bed-ridden by age 26. Fibroblasts showed reduced levels of NDUFA8, as well as other complex I subunits, and reduced complex I activity. The mutation creates an additional Cys residue next to Cys46, which possibly leads to incorrect disulfide bond formation. In addition, Arg47 has multiple interactions within the NDUFA8 subunit, as well as with NDUFA13 and NDUFB5, which would be disrupted in the mutant p.Arg47Cys.

The second individual was compound heterozygous with p.Glu109Lys in NDUFA8 and p.Ala224Val in core subunit NDUFS2 [63]. The transcript for the mutant NDUFA8 subunit was not found, and so the mutation might have affected its stability. Ala224 in the core subunit NDUFS2 is highly conserved and is found near Cys 153 and 160 of a FeS cluster, and so this substitution might also have been deleterious. The infant showed neonatal hypotonia and died at two months.

The third individual was found in a screen of patients with reduced complex I levels [22]. Two potential deleterious mutations were discovered. First was a heterozygous mutation p.Arg135Gln in NDUFA8, and second was a homozygous mutation p.Leu229Pro in C20orf, a spindle assembly factor for microtubles. Since the individual showed decreased levels of complex I activity, the NDUFA8 mutation was implicated, but it was not clear whether the spindle assembly factor had any impact on complex I levels. Arg135 is highly conserved, and it makes an ion pair with Glu59, also a conserved amino acid of NDUFA8. Glu59 is near the disulfide formed by Cys46 and Cys56. This ion pair is exposed on the IMS and does not contact any other subunits.
