*2.1. N-Module Subunits*

*‐*

The first subunits to be described are two subunits that primarily have contacts to core subunits of the N-module (see Supplementary File Table S1 for a Table of Subunit Interactions). Overall, they are rather isolated from other supernumerary subunits, as shown in Figure 2. Beyond those similarities, their properties appear divergent. ‐

‐ **Figure 2.** Location of NDUFA2 and NDUFV3. Most of Complex I is colored gray. Core subunits in the N-module arm are colored light blue (NDUFV1, NDUFV2, and NDUFS1). NDUFA2 and NDUFV3 are shown in ribbons, with NDUFA2 colored purple and NDUFV3 colored red. The two views are rotated 180◦ relative to each other. The structure is from PDB id = 5xtd [9].

#### 2.1.1. NDUFA2

‐ ‐ ‐ ‐ ‐ ‐ The gene for NDUFA2 is located on chromosome 5. The protein contains 99 amino acids with a mass of about 10.9 kDa before the processing of the amino-terminal methionine. This subunit is bound to the matrix arm in a unique fashion, with exclusive contacts to core subunit NDUFS1. It is bound to C-terminal domains of NDUFS1, and it is at least 40 Å from any of the FeS clusters. It has a compact structure that resembles a thioredoxin fold and includes a flat four-stranded beta-sheet (see Figure 3). Two Cys residues, 24 and 58, are found in the loops of the beta-sheet, but they do not appear to be oxidized in the existing structures of mammalian complex I. Contact with NDUFS1 is primarily with the beta-sheet region, including both Cys residues. In culture, NDUFA2 knockout cell lines have been found to lack complex I activity, and an analysis by blue native (BN) gel electrophoresis showed a limited assembly of the N-module—in particular, the loss of core subunits NDUFS1, NDUFV1, and NDUFV2 and supernumerary subunits NDUFS4, NDUFS6, NDUFA7, and NDUFV3. In a cell line with an NDUFA2 knockout, complex I assembly is slightly affected, with a prominent band in BN gel electrophoresis but apparently missing some subunits, probably the N-module. Complex I activity has been found to be extremely low, consistent with a missing NADH binding site [11]. ‐

‐

‐ ‐ ‐ **Figure 3.** Structural features of NDUFA2 from the N-module. The protein is portrayed in a purple-colored ribbon. Sites of mutations are shown in space-filling views: Lys45Thr and Glu57Ala are mitochondrial disease mutations (colored lime). Asn50Asp has been found in breast cancer patients (colored pink). Two Cys residues are colored yellow.

An individual with Leigh syndrome and hypertrophic cardiomyopathy was discovered to be homozygous with a mutation, c.208 + 5 G > A, in the NDUFA2 gene that caused a reduction in correct splicing [12]. Normally, NDUFA2 is coded by three exons, but in this individual, most of the transcripts lacked exon 2, causing a frameshift. No protein or transcript was found, making it a null mutation. The patient died of cardiovascular arrest at eleven months.

‐ Two individuals with leukoencephalopathy were discovered to have mutations in NDUFA2 [13]. One was homozygous for p.Lys45Thr, and the other had compound heterozygous mutations, p.Lys45Thr and a deletion at Asn76, c.225del, and p.Asn76Metfs\*4, thus causing a frameshift and a stop codon after 4 codons. The first was found to have complex I deficiency and lacked an assembled complex I in BN gel electrophoresis. This individual also had a systemic deficiency in carnitine due to a homozygous mutation in SLC22A5. She developed focal epilepsy at six years, was wheelchair-bound at nine years, and was last evaluated at 12 years. The second patient had a similar presentation, including abnormal white matter in the brain. She also had movement difficulties and was last evaluated at age four. Lys45 is found at the interface with NDUFS1 and has close contacts with several NDUFS1 residues, including Gly376, Asp380, and Ser672. It is likely that the binding of NDUFA2 to NDUFS1 is destabilized.

In 2020 [14], another individual was described with a homozygous mutation in NDUFA2, p.Glu57Ala. This residue is at the interface with NDUFS1 and makes a possible ion pair with Arg382 of NDUFS1. It also makes nonbonding contacts with Gly661, Ala662, Asn663, Tyr664, Leu381, Arg382, and Ser383, suggesting a likely disruption of binding. This individual had abnormal white matter in the brain, microcephaly, seizures, and movement disorders. She was last evaluated at four years of age.

In a screen of breast cancer patients [15], a homozygous p.Asp50Asn mutation in NDUFA2 was detected in one individual. This residue is not at the interface with NDUFS1, and so it perhaps does not disrupt the assembly of the enzyme. The significance of this finding is uncertain.
