2.1.2. NDUFV3

The gene for NDUFV3 is found on chromosome 21. This was the first subunit of human complex I known to have multiple isoforms [16–18], which can be found by alternative splicing. It is not an

essential protein, as shown by knockout studies in cell culture [11]. The loss of this gene does not prevent the assembly of complex I, as seen in BN gels, and cells retain the ability to grow in a galactose medium, indicative of oxidative phosphorylation activity. Exogenous NDUFV3 has been seen to exchange with fully assembled complex I, suggesting that it can be incorporated last into the enzyme. The more common isoform has 108 amino acids (74 after the cleavage of the transit peptide) and a mass of 11.8 kDa, while the second isoform has 473 amino acids and a mass of about 51 kDa. Isoform two has an additional exon between the second and the last exons of the shorter form. The additional exon contains a serine-rich region that was found to be phosphorylated [19]. NDUFV3 is not present in fungal complex I from *Yarrowia lipolytica* [7].

NDUFV3 was originally identified in the flavoprotein fraction of bovine heart complex I and now can be seen to have primary contacts with NDUFV1 and NDUFV2 in the human enzyme. It also has limited contact with NDUFS1 and NDUFS4. In the human model of complex I (PDB id = 5xtd) [9], only 33 amino acids are visible, corresponding to residues 74–106. Residues 82–5 are alpha-helical. The longer isoform has an insertion at residue 56 and retains the same C-terminus as isoform one. Therefore, both isoforms can likely bind in the same fashion to other complex I subunits. Both isoforms have been seen in a variety of tissues, but isoform two is more common in cultured cells and in brain tissue [16]. Others have found that isoform one is more prevalent in bovine and murine complex I of heart tissue, as well as that increased Km values for NADH consumption by complex I correlate with an increased extent of the short form in heart tissue [18]. In a cross-linking study of native mouse heart mitochondria [20], both isoforms were identified in cross-links with a surface peptide of malate dehydrogenase, found near the NAD<sup>+</sup> binding site. The binding of malate dehydrogenase and several other enzymes to porcine complex I was demonstrated in 1984 [21]. Other cross-links identified with NDUFV3 were with NDUFV1, NDUFV2, Cox7A1, and Atp5a1—the latter two supporting the proximity of the complexes of oxidative phosphorylation.

Sequence variations have been discovered in NDUFV3 among individuals with decreased levels of complex I activity, but none have been shown to be causative for disease. In one such cohort, four individuals with mutations in NDUFV3 were found [22]. The first individual had p.Arg26Gln, which would occur in the mitochondrial transit peptide and thus might affect import. A second mutation, was found in the same individual in the mitochondrial DNA polymerase G gene POLG, p.Gly11Asp. The second individual had the mutation p.Lys56Asn, which occurs at the splice junction and might affect expression, but this individual also carried a mutation in AMACR, an enzyme associated with branched-chain fatty acid metabolism, p.Val185Ala. The third individual had the mutation p.Gly103Asp, which would occur in both isoforms, in the region that binds to other subunits. Gly103 contacts Trp166 of NDUFS4 in a non-bonding interaction, and it is adjacent to several charged residues. Arg104 of NDUFV3 is near Arg169 of NDUFS4 and Asp426 of core subunit NDUFS1. Glu105 of NDUFV3 and Glu72 of NDUFV2 are nearby. Ser106 is also in this region, and it was found to be phosphorylated in a screen of human cancer cells [23]. Therefore the p.Gly103Asp mutation appears likely to be disruptive to the binding of NDUFV3. The fourth individual had the mutation p.Glu276Lys, which is found only in the long form, near the sites of phosphorylation. This individual also carried a p.Gly154Ser mutation in the core subunit NDUFS8, which is a buried residue, and that substitution is more likely to be deleterious.

#### *2.2. Q-Module Subunits*

NDUFS4, NDUFS6, and NDUFA9 are found in the Q-module, as shown in Figure 4. They interact with each other and with many other subunits in the matrix arm. They appear to have roles in assembling and stabilizing the N- and Q-modules.
