ETFA

The human ETFA gene encodes the Electron-transfer-flavoprotein, alpha subunit, also known as ETF-α.{{cite web | title = Entrez Gene: ETFA electron-transfer-flavoprotein, alpha polypeptide (glutaric aciduria II)| url = https://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2108}} Together with Electron-transfer-flavoprotein, beta subunit, encoded by the 'ETFB' gene, it forms the heterodimeric electron transfer flavoprotein (ETF). The native ETF protein contains one molecule of FAD and one molecule of AMP, respectively.{{cite journal | vauthors = Sato K, Nishina Y, Shiga K | title = Electron-transferring flavoprotein has an AMP-binding site in addition to the FAD-binding site | journal = Journal of Biochemistry | volume = 114 | issue = 2 | pages = 215–22 | date = August 1993 | pmid = 8262902 | doi = 10.1093/oxfordjournals.jbchem.a124157 }}{{cite journal | vauthors = Husain M, Steenkamp DJ | title = Electron transfer flavoprotein from pig liver mitochondria. A simple purification and re-evaluation of some of the molecular properties | journal = The Biochemical Journal | volume = 209 | issue = 2 | pages = 541–5 | date = February 1983 | pmid = 6847633 | doi = 10.1042/bj2090541 | pmc = 1154123 }}

First reports on the ETF protein were based on ETF isolated from porcine liver.{{cite journal | vauthors = Crane FL, Beinert H |title=A Link Between Fatty Acyl CoA Dehydrogenase and Cytochrome C: A New Flavin Enzyme |journal=Journal of the American Chemical Society |date=September 1954 |volume=76 |issue=17 |pages=4491 |doi=10.1021/ja01646a076}}

Porcine and human ETF transfer electrons from mitochondrial matrix flavoenzymes to Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) encoded by the ETFDH gene. ETF-QO subsequently relays the electrons via ubiquinone to complex III in the respiratory chain.{{cite journal | vauthors = Ruzicka FJ, Beinert H | title = A new iron-sulfur flavoprotein of the respiratory chain. A component of the fatty acid beta oxidation pathway | journal = The Journal of Biological Chemistry | volume = 252 | issue = 23 | pages = 8440–5 | date = December 1977 | doi = 10.1016/S0021-9258(19)75238-7 | pmid = 925004 | doi-access = free }} The flavoenzymes that transfer electrons to ETF are involved in fatty acid beta oxidation, amino acid catabolism, choline metabolism, and special metabolic pathways. Defects in either of the ETF subunits or ETFDH cause multiple acyl CoA dehydrogenase deficiency (OMIM # 231680),{{cite web |title=OMIM Entry - # 231680 - MULTIPLE ACYL-CoA DEHYDROGENASE DEFICIENCY; MADD |url=https://www.omim.org/entry/231680 |website=www.omim.org}} earlier called glutaric acidemia type II. MADD is characterized by excretion of a series of substrates of the upstream flavoenzyes, e.g. glutaric, lactic, ethylmalonic, butyric, isobutyric, 2-methyl-butyric, and isovaleric acids.

Evolutionary relationships

ETF is an evolutionarily ancient protein with orthologues found in all kingdoms of life.{{cite journal | vauthors = Toogood HS, Leys D, Scrutton NS | title = Dynamics driving function: new insights from electron transferring flavoproteins and partner complexes | journal = The FEBS Journal | volume = 274 | issue = 21 | pages = 5481–504 | date = November 2007 | pmid = 17941859 | doi = 10.1111/j.1742-4658.2007.06107.x | doi-access = free }} ETFs are grouped into 3 subgroups, I, II, and III. The best studied group are group I ETFs that in eukaryotic cells are localized in the mitochondrial matrix space. Group I ETFs transfer electrons between flavoenzymes. Group II ETFs may also receive electrons from ferredoxin or NADH.

Gene, expression, and subcellular localization

The human ETFA gene encoding the alpha subunit of ETF (ETF-α) is localized on chromosome 15 (15q24.2-q24.3). It is composed of 12 exons. Little is known about its promoter and transcriptional regulation. Global expression analyses show that it is expressed at substantial levels in most tissues ([https://www.proteomicsdb.org/proteomicsdb/#protein/proteinDetails/52989/expression PROTEOMICXS DB]). ETF-α is translated as a precursor protein with an N-terminal mitochondrial targeting sequence.{{cite journal | vauthors = Ikeda Y, Keese SM, Tanaka K | title = Biosynthesis of electron transfer flavoprotein in a cell-free system and in cultured human fibroblasts. Defect in the alpha subunit synthesis is a primary lesion in glutaric aciduria type II | journal = The Journal of Clinical Investigation | volume = 78 | issue = 4 | pages = 997–1002 | date = October 1986 | pmid = 3760196 | doi = 10.1172/JCI112691 | pmc = 423742 }} It is posttranslationally imported into the mitochondrial matrix space, where the targeting sequence is cut off.

Posttranslational modifications and regulation

Acetylation and succinylation of lysine residues and phosphorylation of serine and threonine residues in ETF-α have been reported in mass spectrometric analyses of posttranslational modifications [https://www.uniprot.org/uniprot/P13804 P13804]. Electron transfer flavoprotein regulatory factor 1 (ETFRF1) has been identified as a protein that specifically binds ETF and this interaction has been indicated to inactivate ETF by displacing the FAD.{{cite journal | vauthors = Floyd BJ, Wilkerson EM, Veling MT, Minogue CE, Xia C, Beebe ET, Wrobel RL, Cho H, Kremer LS, Alston CL, Gromek KA, Dolan BK, Ulbrich A, Stefely JA, Bohl SL, Werner KM, Jochem A, Westphall MS, Rensvold JW, Taylor RW, Prokisch H, Kim JP, Coon JJ, Pagliarini DJ | display-authors = 6 | title = Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function | journal = Molecular Cell | volume = 63 | issue = 4 | pages = 621–632 | date = August 2016 | pmid = 27499296 | doi = 10.1016/j.molcel.2016.06.033 | pmc = 4992456 }}

Structure and interaction with redox partners

As first shown for porcine ETF, one chain of ETF-α assembles with one chain of ETF-β, and one molecule each of FAD and AMP to the dimeric native enzyme.{{cite journal | vauthors = Hall CL, Kamin H | title = The purification and some properties of electron transfer flavoprotein and general fatty acyl coenzyme A dehydrogenase from pig liver mitochondria | journal = The Journal of Biological Chemistry | volume = 250 | issue = 9 | pages = 3476–86 | date = May 1975 | doi = 10.1016/S0021-9258(19)41540-8 | pmid = 1168197 | doi-access = free }}{{cite journal | vauthors = Gorelick RJ, Mizzer JP, Thorpe C | title = Purification and properties of electron-transferring flavoprotein from pig kidney | journal = Biochemistry | volume = 21 | issue = 26 | pages = 6936–42 | date = December 1982 | pmid = 7159575 | doi = 10.1021/bi00269a049 }}{{cite journal | vauthors = Sato K, Nishina Y, Shiga K | title = In vitro refolding and unfolding of subunits of electron-transferring flavoprotein: characterization of the folding intermediates and the effects of FAD and AMP on the folding reaction | journal = Journal of Biochemistry | volume = 120 | issue = 2 | pages = 276–85 | date = August 1996 | pmid = 8889811 | doi = 10.1093/oxfordjournals.jbchem.a021410 }} The crystal structure of human ETF was reported in 1996.{{cite journal | vauthors = Roberts DL, Frerman FE, Kim JJ | title = Three-dimensional structure of human electron transfer flavoprotein to 2.1-A resolution | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 93 | issue = 25 | pages = 14355–60 | date = December 1996 | pmid = 8962055 | doi = 10.1073/pnas.93.25.14355 | pmc = 26136 | doi-access = free }} This showed that ETF consists of three distinct domains (I, II, and III). The FAD is bound in a cleft between the two subunits and interacts mainly with the C-terminal part of ETF-α. The AMP is buried in domain III. A crystal structure of the complex of one of its interactors, medium-chain acyl-CoA dehydrogenase (MCAD; gene name ACADM) has been determined.{{cite journal | vauthors = Toogood HS, van Thiel A, Basran J, Sutcliffe MJ, Scrutton NS, Leys D | s2cid = 6901700 | title = Extensive domain motion and electron transfer in the human electron transferring flavoprotein.medium chain Acyl-CoA dehydrogenase complex | journal = The Journal of Biological Chemistry | volume = 279 | issue = 31 | pages = 32904–12 | date = July 2004 | pmid = 15159392 | doi = 10.1074/jbc.M404884200 | doi-access = free }} (toogood 2004+2007). This identified a so-called recognition loop formed by ETF-β that anchors ETF on one subunit of the homotetrameric MCAD enzyme. This interaction triggers conformational changes and the highly mobile redox active FAD domain of ETF swings to the FAD domain of a neighboring subunit of the MCAD tetramer bringing the two FAD molecules into close contact for interprotein electron transfer.

Molecular Function

Human ETF receives electrons from at least 14 flavoenzymes and transfers them to ETF-ubiquinone oxidoreductases (ETF:QO) in the inner mitochondrial membrane. ETF:QO in turn relays them to ubiquinone from where they enter the respiratory chain at complex III. Most of the flavoenzymes transferring electrons to ETF are participating in fatty acid oxidation, amino acid catabolism, and choline metabolism. ETF and ETF:QO thus represent an important hub for transfer of electrons from various redox reactions and feeding them into the respiratory chain for energy production.

Genetic deficiencies and molecular pathogenesis

Deleterious mutations in the ETFA and ETFB genes encoding ETF or the ETFDH gene encoding ETF:QO are associated with multiple acyl-CoA dehydrogenase deficiency (MADD; [https://www.omim.org/entry/231680 OMIM #231680]; previously called glutaric aciduria type II).{{cite journal | vauthors = Prasun P | title = Multiple Acyl-CoA Dehydrogenase Deficiency | date = 1993 | pmid = 32550677 | veditors = Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJ, Stephens K, Amemiya A }} Biochemically, MADD is characterized by elevated levels of a series of carnitine conjugates of the substrates of the different partner dehydrogenases of the ETF/ETF:QO hub, e.g. glutaric, lactic, ethylmalonic, butyric, isobutyric, 2-methyl-butyric, and isovaleric acids.[5] Accumulation of substrates and derivatives of the upstream dehydrogenases and energy deficiency upon fasting cause the clinical phenotype. Mostly depending on the severity of the mutation, the disease is divided into three subgroups: type I (neonatal onset with congenital anomalies), type II (neonatal onset without congenital anomalies), and type III (late onset). There is no cure for the disease, and treatment is employing a diet limiting protein and fat intake, avoidance of prolonged fasting, both to alleviate the flow through the partner dehydrogenases. In addition, supplementation of riboflavin, the precursor of the FAD co-factor can stabilize mutant ETF and ETF:QO variants with certain missense mutations.{{cite journal | vauthors = Henriques BJ, Olsen RK, Bross P, Gomes CM | title = Emerging roles for riboflavin in functional rescue of mitochondrial β-oxidation flavoenzymes | journal = Current Medicinal Chemistry | volume = 17 | issue = 32 | pages = 3842–54 | date = 2010 | pmid = 20858216 | doi = 10.2174/092986710793205462 }}{{cite journal | vauthors = Henriques BJ, Bross P, Gomes CM | title = Mutational hotspots in electron transfer flavoprotein underlie defective folding and function in multiple acyl-CoA dehydrogenase deficiency | journal = Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease | volume = 1802 | issue = 11 | pages = 1070–7 | date = November 2010 | pmid = 20674745 | doi = 10.1016/j.bbadis.2010.07.015 | s2cid = 12147360 | url = https://hal.archives-ouvertes.fr/hal-00623296/file/PEER_stage2_10.1016%252Fj.bbadis.2010.07.015.pdf }}