life Review Analysis of Human Mutations in the Supernumerary Subunits of Complex I Quynh-Chi L. Dang, Duong H. Phan, Abigail N. Johnson, Mukund Pasapuleti, Hind A. Alkhaldi, Fang Zhang and Steven B. Vik * Department of Biological Sciences, Southern Methodist University, Dallas, TX 75287, USA; [email protected] (Q.-C.L.D.); [email protected] (D.H.P.); [email protected] (A.N.J.); [email protected] (M.P.); [email protected] (H.A.A.); [email protected] (F.Z.) * Correspondence: [email protected] Received: 30 October 2020; Accepted: 16 November 2020; Published: 20 November 2020 Abstract: Complex I is the largest member of the electron transport chain in human mitochondria. It comprises 45 subunits and requires at least 15 assembly factors. The subunits can be divided into 14 “core” subunits that carry out oxidation–reduction reactions and proton translocation, as well as 31 additional supernumerary (or accessory) subunits whose functions are less well known. Diminished levels of complex I activity are seen in many mitochondrial disease states. This review seeks to tabulate mutations in the supernumerary subunits of humans that appear to cause disease. Mutations in 20 of the supernumerary subunits have been identified. The mutations were analyzed in light of the tertiary and quaternary structure of human complex I (PDB id = 5xtd). Mutations were found that might disrupt the folding of that subunit or that would weaken binding to another subunit. In some cases, it appeared that no protein was made or, at least, could not be detected. A very common outcome is the lack of assembly of complex I when supernumerary subunits are mutated or missing. We suggest that poor assembly is the result of disrupting the large network of subunit interactions that the supernumerary subunits typically engage in. Keywords: mitochondria; mammalian complex I; NADH dehydrogenase; complex I assembly; complex I structure; complex I deficiency; supernumerary subunits; electron transport chain; mitochondrial dysfunction; Leigh syndrome 1. Introduction Mutations in the genes that encode complex I are responsible for a large fraction of all mitochondrial diseases. For example, 20–30% of cases in childhood mitochondrial disease (MD) are related to complex I dysfunction [1,2]. There seem to be many reasons for this. Complex I genes make up a large component of the mitochondrial and nuclear genome. Complex I is encoded by seven mitochondrial genes (out of 13 total) and 37 nuclear genes. There are also at least 15 complex I assembly factors [3]. Mitochondrial DNA (mtDNA) is especially prone to mutation due to insufficient DNA repair systems. All of the various functions of complex I can be impacted by mutation. Complex I forms supercomplexes with other members of the respiratory chain, such as complex III (cytochrome bc1) and complex IV (cytochrome c oxidase). It is metabolically linked to the citric acid cycle by NADH and to ATP synthesis by proton translocation. Furthermore, it is a major site of superoxide generation in mitochondria. Therefore, mutations that alter or degrade complex I function will typically have wider effects on mitochondrial function. Complex I is a boot-shaped multi-subunit enzyme embedded in the inner mitochondrial membrane (see Figure1). Its primary role is to oxidize NADH while reducing ubiquinone and translocating protons across the membrane. One arm extends into the matrix space, and it contains the flavin Life 2020, 10, 296; doi:10.3390/life10110296 www.mdpi.com/journal/life Life 2020, 10, 296 2 of 35 Life 2020, 10, x FOR PEER REVIEW 2 of 35 mononucleotide (FMN) and all of the iron–sulfur (FeS) clusters necessary for electron transfer to ubiquinone.the flavin The mononucleotide membrane (FMN) arm contains and all of subunits the iron–sulfur that translocate (FeS) clusters the necessary protons from for electron the matrix spacetransfer to the intermembraneto ubiquinone. The space membrane (IMS). arm These contains core functionssubunits that are translocate carried out the by protons the fourteen from the “core” subunitsmatrix that space appear to the inintermembrane all known examples space (IMS). of These complex core functions I, including are carried bacteria. out by Seven the fourteen of the core subunits“core” are subunits membrane-embedded, that appear in all known and theseexamples seven of complex are all encodedI, including in bacteria. mtDNA: Seven ND1, of the ND2, core ND3, ND4,subunits ND4L, ND5,are membrane and ND6.‐embedded, The other and seven these core seven subunits are all encoded are found in inmtDNA: the matrix ND1, arm ND2, and ND3, contain ND4, ND4L, ND5, and ND6. The other seven core subunits are found in the matrix arm and contain the FMN and all of the FeS clusters. NDUFV1 contains the flavin and one tetranuclear FeS cluster, the FMN and all of the FeS clusters. NDUFV1 contains the flavin and one tetranuclear FeS cluster, N3. NDUFV2 contains one binuclear FeS cluster N1a, which is not on the main electron transfer N3. NDUFV2 contains one binuclear FeS cluster N1a, which is not on the main electron transfer path. path.NDUFS1 NDUFS1 contains contains one onebinuclear binuclear FeS cluster, FeS cluster, N1b, and N1b, two tetranuclear and two tetranuclear clusters, N4 clusters,and N5. NDUFS8 N4 and N5. NDUFS8contains contains two ferredoxin two ferredoxin-like‐like clusters, clusters, N6a and N6a N6b. and NDUFS7 N6b. NDUFS7 contains contains the tetranuclear the tetranuclear cluster N2, cluster N2, whichwhich is proximal to to the the ubiquinone ubiquinone binding binding site. site.For recent For recent reviews reviews of complex of complex I, see [4–7]; I, see for [ 4–7]; for supernumerarysupernumerary subunits, see see [8]. [8 ]. FigureFigure 1. Structure1. Structure of of complex complex I with supernumerary supernumerary subunits subunits highlighted. highlighted. Core subunits Core subunits in the in the matrixmatrix armarm areare coloredcolored light light blue. blue. Core Core subunits subunits in inthe themembrane membrane arm armare arecolored colored beige. beige. SupernumerarySupernumerary subunits subunits that arethat described are described in this reviewin this are review colored are and colored labeled. and Other labeled. supernumerary Other subunitssupernumerary are white. subunits The two are views white. are The rotated two views 180◦ arerelative rotated to 180 each˚ relative other. to The each structure other. The is from the Proteinstructure Data is from Bank the file Protein 5xtd Data (PDB Bank id =file5xtd) 5xtd [9(PDB]. All id structural= 5xtd) [9]. imagesAll structural were generatedimages were using Jmolgenerated (http://www.jmol.org using Jmol (http://www.jmol.org).). The remainingThe remaining thirty-one thirty‐one subunits subunits (one (one is is foundfound in in two two copies) copies) are are supernumerary supernumerary (or accessory) (or accessory) subunits,subunits, and muchand much less less is known is known about about their their functions. functions. They They are are typically typically much much smallersmaller thanthan the core core subunits, and they are distributed on all surfaces of complex I. Some cross the membrane, while subunits, and they are distributed on all surfaces of complex I. Some cross the membrane, while others others are localized to the matrix face or the IMS. The naming of these subunits has generally followed are localized to the matrix face or the IMS. The naming of these subunits has generally followed their their co‐purification with various fractions of complex I: FV for the flavoprotein fraction, FS for the co-purificationFeS protein withfraction, various FA for fractions the alpha of fraction complex associated I: FV for with the the flavoprotein matrix arm subunits, fraction, and FS forFB for the FeS proteinthe fraction, beta fraction FA forassociated the alpha with fraction the membrane associated proteins. with The the exception matrix arm is NDUFAB1, subunits, andthe acyl FB forcarrier the beta fractionprotein, associated which resembles with the membranean enzyme in proteins. lipid biosynthesis. The exception This subunit is NDUFAB1, appears theto have acyl an carrier essential protein, whichrole resembles apart from an complex enzyme I, in and lipid it is biosynthesis. the only protein This that subunit appears appears in two copies. to have an essential role apart from complexFrom I,an and evolutionary it is the only point protein of view, that the core appears subunits in two can copies. be organized into three modules. The FromN‐module an evolutionary contains NDUFV1, point NDUFV2, of view, and the NDUFS1, core subunits and it is can defined be organized by the source into of electrons three modules. to The N-modulethe complex, contains the substrate NDUFV1, NADH. NDUFV2, This module and NDUFS1,is related to and various it is defined NAD‐linked by the dehydrogenases. source of electrons The Q‐module contains the remaining peripheral subunits NDUFS2, NDUFS3, NDUFS7, and to the complex, the substrate NADH. This module is related to various NAD-linked dehydrogenases. NDUFS8, as well as two membrane subunits, ND1 and ND3, that contain the remaining FeS clusters The Q-module contains the remaining peripheral subunits NDUFS2, NDUFS3, NDUFS7, and NDUFS8, and contribute to the ubiquinone binding site. This module is related to various membrane‐bound as wellhydrogenases. as two membrane Finally, the subunits, remaining ND1 membrane and ND3, subunits, that contain ND2, ND4, the remaining ND4L, ND5, FeS and clusters ND6 and contributecompose to the the P‐ ubiquinonemodule (for proton binding translocating) site. This and module are related is related to subunits to various of an Na+ membrane-bound/H+ antiporter, hydrogenases. Finally, the remaining membrane subunits, ND2, ND4, ND4L, ND5, and ND6 compose the P-module (for proton translocating) and are related to subunits of an Na+/H+ antiporter, the Mrp complex [10]. This grouping of subunits also corresponds to the assembly pathway. Q-module subunits Life 2020, 10, 296 3 of 35 appear to assemble first, followed by the stepwise addition of the P-module, associated with the three major subunits ND2, ND4, and ND5.