*2.8. Subunits from the ND5-Module*

Finally, three subunits are next described from the ND5-module: NDUFB3, NDUFB6, and NDUFB8, as shown in Figure 16. These subunits do not interact directly with each other, but they serve to enclose the distal end of complex I on all sides. In particular, NDUFB8 clamps the lateral helix of core subunit ND5.

*‐*

‐

‐ **Figure 16.** Location of NDUFB3, NDUFB6, and NDUFB8. Most of Complex I is colored gray. Core subunits in the ND5-module are colored light blue (ND5). NDUFB3, NDUFB6, and NDUFB8 are shown in ribbons, with NDUFB3 colored violet, NDUFB6 colored salmon, and NDUFB8 colored pink. The two views are rotated 180◦ relative to each other. The structure is from PDB id = 5xtd [9].

#### 2.8.1. NDUFB3

‐ ‐ ‐ ‐ ‐ ‐ The gene for NDUFB3 is found on chromosome 2. The encoded protein has 99 amino acids with a mass of about 11.4 kDa. The N-terminal Met appears to be cleaved. NDUFB3 is found at the distal end of complex I near core subunit ND5. It has a small N-terminal domain composed of three alpha-helices on the matrix side, and the C-terminus forms an alpha-helix, residues 62–89, that crosses the membrane. NDUFB3 contacts NDUFB9 and NDUFAB1 on the matrix side, as well as NDUFB2 and core subunit ND5, primarily in the membrane region. In knockout cell lines, the absence of NDUFB3 was found to result in the near total loss of complex I assembly and a reduction in the level of subunits from both Nand ND5 assembly modules [11].

‐ ‐ ‐ ‐ Two mutations have been identified in the gene for NDUFB3 among over 10 individuals. In a report from [78], p.Trp22Arg appeared as a homozygous mutation in a girl who exhibited intrauterine growth retardation and premature birth at 31 weeks. She died at four months of age. Very low levels of complex I activity (<15%) were measured in fibroblasts, and this phenotype was rescued by lentivector complementation. A second individual was identified with compound heterozygous mutations: p.Trp22Arg and p.Gly70X [79]. The level of complex I activity was <25% of normal, and the individual exhibited muscular hypotonia, developmental delay, and lactic acidosis. In fibroblasts, complex I activity could be recovered by the ectopic expression of wild-type NDUFB3, though not either mutant form. The health status of this individual was not reported. In contrast, two more recent reports have identified the p.Trp22Arg mutation in older and seemingly healthier individuals. Ten children, up to 10 years of age, were identified as homozygous carriers of p.Trp22Arg in NDUFB3 [80]. These individuals with short statures and prominent foreheads ranged from 10 months to 10 years in age, and their complex I activity levels ranged from 25 to 35% of normal levels. Various levels of assembled complex I were seen from these individuals using blue native gel electrophoresis. More recently, a 32-year-old previously diagnosed with non-alcoholic steatohepatitis was discovered to be homozygous p.Trp22Arg [81]. This individual had reduced levels of complex I activity (<30% of normal) and suffered from oculomotor dysfunction with optic nerve anomalies, episodes of lactic acidosis during surgical interventions, and progressive fatigue.

The latter two studies emphasized that the context of these two mutations in NDUFB3 appears to be significant. The level of complex I activity, and its assembly, is likely on the borderline between tolerable and deleterious. Trp22 is found in a conserved region of NDUFB3 on the matrix side of the membrane. The NH side chain of Trp is in position to H-bond to the OH side chain of Tyr85 of NDUFAB1. It also makes nonbonding interactions with Arg45 and Gln46 of NDUFB2. Therefore, the mutation to the positively charged Arg is likely to be disruptive. The second mutation, p.Gly80X,

occurs in the last exon and so is not likely to trigger nonsense-mediated decay. This residue occurs in the transmembrane region, and the stop codon would truncate the protein and eliminate the transmembrane helix. Gly70 is flanked by Lys69 and Lys72, which probably demarcates the matrix end of the lipid bilayer. Thus, it seems possible that the truncated NDUFB3 would retain some ability to assemble and provide normal interactions on the matrix side.
