2.4.1. NDUFA6

‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ The gene for NDUFA6 is located on chromosome 22. The encoded protein has 128 amino acids, with a mass of about 15.1 kDa. This subunit is found on the matrix arm. Its tertiary structure resembles a four-helix bundle in which the fourth helix is pulled away from the other three (see Figure 9a). Helices 1 and 2 interact with one of the two NDUFAB1 subunits, which are also known as the acyl carrier proteins and presumed to be involved in fatty acid biosynthesis. The short C-terminal helix interacts with core subunit NDUFS1, while core subunit NDUFS3 and supernumerary subunit NDUFA9 interact with both the N-terminal helices and the extended C-terminus. Thus, NDUFA6 bridges the N- and Q-modules. NDUFA6 is a member of the LYR family [56], whose members are known to interact with large mitochondrial complexes. Leu35 of the LYR motif fits inside the bundle of three helices, while Tyr36 and Arg37 contact NDUFAB1. In knockout cell lines, the assembly of full-size complex I was diminished, as seen in BN gel electrophoresis, and the levels of all subunits associated with the N-module of the peripheral arm were decreased by two-fold [11]. ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐

**Figure 9.** Structural features of the LYR proteins, NDUFA6 and NDUFB9. (**a**) NDUFA6 (colored yellow) is shown with its partner NDUFAB1 (colored gray), known as one of the two acyl carrier proteins (ACPs). NDUFA6 binds the 4′ -phosphopantethiene (4′ -PPT) analog substrate for the ACP. Tyr36 and Arg37 of the LYR motif are shown in space-filling and colored cyan. The site of the Arg64Pro mutation is shown in space-filling and colored blue. (**b**) NDUFB9 (colored lime) is shown with its partner NDUFAB1 (colored gray), the other one of the two ACPs. NDUFB9 binds the 4′ -PPT analog substrate for the ACP. Tyr20 and Lys21 of the LYR motif are shown in space-filling and colored pink. The site of the Arg64Pro mutation is shown in space-filling and colored magenta.

Mutations in NDUFA6 have been described for four individuals, though only recently [57]. The most severe of the illnesses occurred in an individual with compound heterozygous mutations: p.Arg64Pro and c.265 G > T, a nonsense mutation at Glu89. An analysis of tissue demonstrated almost no fully assembled complex I and almost no in-gel activity in BN gel electrophoresis. This individual died after about two days. Tissue from two other individuals was analyzed and showed low levels of assembled complex I with activity in BN gel electrophoresis. Residue Arg64 is ion-paired with Asp111 of NDUFAB1 and is near Asp92 of NDUFAB1 and Tyr36 of the LYR motif of NDUFA6. The adjacent Ser112 of NDUFAB1 is part of the catalytic site of the acyl carrier enzyme and is bound by a substrate in the human structure (PDB id = 5xtd) [9].

The second individual was homozygous for a two-nucleotide deletion at Glu111, c.331\_332del, resulting in 35 altered amino acids before a stop codon appeared [57]. This transcript was found at normal levels, thus indicating the lack of nonsense-mediated decay, presumably because it occurs in the last of three exons near the normal termination codon. If this transcript was translated, the protein would lack the C-terminal alpha-helix that interacts with core subunit NDUFS1. The third individual was homozygous for a c.3 G > A, substitution in the start codon. It was suggested that a transcript starting from an upstream Met codon, as predicted by another isoform, might have permitted some expression of a functional protein. Both individuals showed low levels of fully assembled complex I with activity, and their outcomes were only somewhat better than the first, with abnormal white matter in the brain, a loss of movement and vision, and seizures. The fourth individual was compound heterozygous with two frameshift mutations: p.Met104Cysfs\*35 and p.Leu119Tyrfs\*20. This child died in infancy, and no biochemical analyses were performed.
