*2.7. Subunits Form the ND4-Module*

Two subunits from the ND4-module are described next: NDUFA11 and NDUFB11, as shown in Figure 14. Both are membrane proteins but are found on alternate sides of ND4 and do not contact each other.

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‐ **Figure 14.** Location of NDUFA11 and NDUFB11. Most of Complex I is colored gray. Core subunits in the ND4-module are colored light blue (ND4). NDUFA11 and NDUFB11are shown in ribbons, NDUFA11 colored blue and NDUFB11 colored purple. The two views are rotated 180◦ relative to each other. The structure is from PDB id = 5xtd [9].

#### 2.7.1. NDUFA11

‐ The NDUFA11 gene is located on chromosome 19. NDUFA11 is a 14.7 kDa protein with four transmembrane helices and little exposure outside the membrane. NDUFA11 has two isoforms. The first isoform contains 140 amino acids after the cleavage of the initiator methionine and is N-acetylated at Ser2. It consists of five alpha helices: a short helix is found on the IMS side, followed by membrane spanning helices 3–11, 17–45, 48–82, 87–106, and 107–137. NDUFA11 contacts the core subunits ND2, ND4, and ND5, as well as the supernumerary subunit NDUFB5. The second isoform is rare and contains 228 amino acids. In a previous study, it was found that the knockdown of NDUFA11 by RNA interference (siRNA) in human cell culture led to partially assembled subcomplexes visualized by blue native gels [71]. It was concluded that NDUFA11 acts like an assembly factor in complex I. Furthermore, it was shown that, in a knockout strain of cultured human cells, the loss of NDUFA11 resulted in no expression of complex I in BN gel electrophoresis [11].

‐ ‐ ‐ ‐ The first identified NDUFA11 clinical mutation, a G to A mutation at the exon-intron junction (exon 1-IVS1) donor splice site, c.99+5 G > A, was identified in 2008 [72]. Six patients from three unrelated families were products of consanguineous marriages and were found to be homozygous for the mutation. They were clinically affected with either fatal infantile lactic acidemia or encephalocardiomyopathy. Though heterozygotes were identified in two families, all family members besides the patients were healthy. The patient from Family A and the two patients from Family B had similar clinical presentations. They all developed severe metabolic acidosis and hyperlactatemia within 10–24 h of age. All three patients from Families A and B died from acidosis within 6–40 days of age. In Family C, three patients were homozygous for this splice mutation. They experienced slow psychomotor development, hypertrophy of myocardial walls, acidosis, and generalized brain atrophy. Two out of the three patients from Family C died at 18 months and four years of age, but the third patient from this family lived until at least six months of age. The presence of both wild-type and mutant mRNA transcripts in the patients' fibroblasts indicates that the varied clinical presentation may have been caused by variable splicing that produced variable mutant/wild-type transcript ratios. They suggested that normal splicing would have produced a protein that was responsible for the detected activity, while alternative splicing would have yielded a nonfunctional protein.

In 2019, a two more NDUFA11 clinical mutations, p.Ala132Pro and p.Thr106Ile, were found in isoform two of NDUFA11 [73]. These mutations were found simultaneously in a compound heterozygote patient with mitochondrial myopathy. The patient developed late-onset symptoms of a neuromuscular disorder, bilateral hearing loss, saccadic eye movements, and proximal leg weakness. The good health of the patient's offspring and the absence of neuromuscular disease in the patient's family history suggested that these two mutations were spontaneous. In contrast to isoform one, NDUFA11 isoform two is rare and appears in skeletal muscle. Thus, a mutation in isoform two may

only lead to mild muscular impairment. The structure of this rare isoform of NDUFA11 has not yet been determined, so we cannot confirm that the mutation causes a structural disruption within this subunit.

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