2.2.1. NDUFS4

The gene for NDUFS4 is found on chromosome 5. NDUFS4 is a protein that was initially found in the iron–sulfur protein fraction of bovine heart complex I. It is synthesized as 175 amino acids, and after the cleavage of the mitochondrial transit peptide, the mature protein is 133 amino acids with a mass of about 15.3 kDa. As shown in Figure 1, it is located in the matrix arm of complex I and contacts core subunits NDUFS1, NDUFS3, NDUFS8, and NDUFV1, as well as supernumerary subunits NDUFA6 and NDUFA9. It fits between two lobes of core subunit NDUFS1. It can be understood as a globular protein with a mixed alpha/beta architecture and three extensions that reach towards the chain of FeS clusters (see Figure 5). The C-terminus, in particular, is within about 9 Å of FeS clusters N3 and N1b, found in NDUFV1 and NDUFS1, respectively. A loop ending in Met87 is within 10 Å of FeS clusters N4 and N5 of NDUFS1, and a second loop including Met112 is pointing toward the last three FeS clusters—N6a, N6b, and N2—but is at least 13 Å away. NDUFS4 has been found to be phosphorylated at residue Ser173, near the C-terminus [24]. It has been suggested that this improves the net import of the subunit by inhibiting its return to the cytoplasm. In a knockout cell line of cultured human cells, the loss of NDUFS4 had a mild effect on assembly of complex I, causing it to migrate as a smaller-than-normal-sized complex in BN gel electrophoresis, probably lacking the N-module [11]. ‐

‐ **Figure 4.** Location of NDUFS4, NDUFS6, and NDUFA9. Most of Complex I is colored gray. Core subunits in the Q-module are colored light blue (NDUFS2, NDUFS3, NDUFS7, and NDUFS8). NDUFS4, NDUFS6, and NDUFA9 are shown in ribbons, with NDUFS4 colored magenta, NDUFS6 colored green, and NDUFA9 colored cyan. The two views are rotated 180◦ relative to each other. The structure is from PDB id = 5xtd [9]. ‐ ‐ ‐ ‐ ‐

**Figure 5.** Structural features of 3 interacting subunits from the Q-module. The proteins are portrayed in ribbons. NDUFS4 is colored magenta. The sites of two mutations in this subunit, Trp114Arg and Asp119His, are shown in space-filling and are colored blue. NDUFS6 is colored green. The zinc bind residues are shown in space-filling, with Cys colored yellow and His colored green. The site of the mutation, Cys115Tyr is colored black. NDUFA9 is colored cyan, and its bound ligand NADP<sup>+</sup> is shown in space-filling with CPK colors (e.g., carbon gray, nitrogen blue, oxygen red, phosphorus orange). The sites of 2 mutations, Arg321Pro and Arg360Cys, are shown in space-filling and are colored red.

Three human mutations in NDUFS4 from Leigh syndrome patients were examined biochemically [25]: a duplication of AAGTC at position 466–470 of the coding sequence, a single G deletion at position 289 (in Trp97), and a nonsense mutation, c.44 G > A, p.Trp15X, in the first exon of the gene. Nonsense-mediated decay apparently eliminated the transcript in the second case. In the third case, c.44 G > A, it was later determined that three alternative splice variants were produced [26]. In all three cases, the mutations were homozygous, and it was demonstrated that little or no protein was made. An analysis of these null mutants from cultured fibroblasts showed that complex I did not assemble by BN gel electrophoresis and that little or no activity could be measured [25].

Two clinical missense mutations have been identified in this subunit, p.Trp114Arg [27] and p.Asp119His [28], both resulting in Leigh syndrome. Several frameshift mutations have been identified, including the homozygous c.221delC (p.Thr74Ifs\*17) and the compound heterozygous mutations c.462delA (p.Lys154Nfs\*34) and c.99-1 G > A) (p.Ser34Ifs\*4), which also appear to cause Leigh syndrome [22]. Both missense mutations are found in the loop nearest to the N2/N6ab FeS clusters. Perhaps more importantly, this extension of NDFS4 is part of a junction of three subunits, with NDUFA9, the NADP<sup>+</sup> binding protein, and an extension of core subunit NDUFS3. The individual with the p.Trp114Arg mutation was found to be homozygous for this allele, while both parents were found to be heterozygous [27]. This individual was diagnosed with myocarditis, respiratory failure, delirium, and basal ganglia abnormalities in the brain as seen by magnetic resonance imaging (MRI), and she was found to have complex I deficiency. Trp114 is a conserved residue that has close contact with Gln228 of NDUFS3 and is near several residues of core subunit NDUFS8. In the human structure (PDB id = 5xtd), the glutamine oxygen from the sidechain is pointing into the tryptophan ring, but it seems that the amino group would more logically assume that position. In conclusion, it seems likely that the p.Trp114Arg mutation would be disruptive and might impact the transfer of electrons through complex I.

The individual with the p.Asp119His mutation was compound heterozygous, with a second allele containing a Lys154 frame shift mutation, and he died in his third year [28]. While these mutations were found in a heterozygous situation (mother, father, and brother), there was no clinical presentation, suggesting that both mutations contributed to declining health. In a different study, the transcript for the frameshift allele was shown to exist, indicating that nonsense-mediated decay did not occur and suggesting that the protein was made. Asp119 is primarily contacted by other residues in NDUFS4, including an ion pairing with Arg75. The closest contact with subunit NDUFA9 is residue Lys45 at a distance of over 5 Å. Therefore, it is likely that the Asp119His substitution would be deleterious. If we assume that the frameshift mutation would produce a protein that is truncated at residue 154, that would remove a small but highly conserved domain that normally interacts with NDUFV3, and with core subunits NDUFV1, NDUFV2, and NDUFS1, in the vicinity of the FeS clusters N3 and N1b. Even this loss of about 20 amino acids would appear to be disruptive, and it seems possible that the truncated protein might be found at lower-than-normal levels due to instability.
