Electron Transfer Flavoprotein Dehydrogenase Research Articles

M.P.3.03 Treatment of myopathic form of coenzyme Q10 deficiency caused by electron-transferring-flavoprotein dehydrogenase ( ETFDH) gene mutations

Primary or secondary Coenzyme Q10 (CoQ10) deficiency in muscle has been demonstrated in various phenotypes and response to CoQ10 supplementation has shown benefit in most cases. We have recently identified a group of patients with isolated myopathic form of CoQ10 deficiency and mutations in electron-transferring-flavoprotein dehydrogenase (ETFDH) gene. This gene is also responsible from glutaric aciduria type II (GAII), a disorder known for good response to riboflavin treatment. We discuss treatment strategy with CoQ10 and riboflavin in this disorder.

M.O.2 Mutations in the electron-transferring-flavoprotein dehydrogenase ( ETFDH) gene cause myopathic form of coenzyme Q10 deficiency

Coenzyme Q10 (CoQ10) deficiency has been identified in different clinical settings with various genetic background. We report patients having an isolated myopathic phenotype and mutations in a previously unreported gene to cause CoQ10 deficiency.

M.P.4.17 Late onset multiple acyl-CoA dehydrogenase deficiency (MADD) associated with novel ETFDH mutations in two Chinese patients

Multiple acyl-CoA dehydrogenase deficiency, also known as glutaric aciduria type II, is a disorder of fatty acid, amino acid, and choline metabolism with autosomal recessive inheritance. The disorder may be due to defect(s) in the alpha- or beta-subunit of electron transfer flavoprotein (ETFA, ETFB) or ETF dehydrogenase (ETFDH). Unlike MADD with neonatal onset, late onset MADD has highly variable presentation and is more rare, with only nine cases with identified genotypes reported to date. We report two unrelated Chinese patients with late onset MADD.

The myopathic form of coenzyme Q10 deficiency is caused by mutations in the electron-transferring-flavoprotein dehydrogenase (ETFDH) gene

Coenzyme Q10 (CoQ10) deficiency is an autosomal recessive disorder with heterogenous phenotypic manifestations and genetic background. We describe seven patients from five independent families with an isolated myopathic phenotype of CoQ10 deficiency. The clinical, histological and biochemical presentation of our patients was very homogenous. All patients presented with exercise intolerance, fatigue, proximal myopathy and high serum CK. Muscle histology showed lipid accumulation and subtle signs of mitochondrial myopathy. Biochemical measurement of muscle homogenates showed severely decreased activities of respiratory chain complexes I and II + III, while complex IV (COX) was moderately decreased. CoQ10 was significantly decreased in the skeletal muscle of all patients. Tandem mass spectrometry detected multiple acyl-CoA deficiency, leading to the analysis of the electron-transferring-flavoprotein dehydrogenase (ETFDH) gene, previously shown to result in another metabolic disorder, glutaric aciduria type II (GAII). All of our patients carried autosomal recessive mutations in ETFDH, suggesting that ETFDH deficiency leads to a secondary CoQ10 deficiency. Our results indicate that the late-onset form of GAII and the myopathic form of CoQ10 deficiency are allelic diseases. Since this condition is treatable, correct diagnosis is of the utmost importance and should be considered both in children and in adults. We suggest to give patients both CoQ10 and riboflavin supplementation, especially for long-term treatment.

Identification of the human mitochondrial FAD transporter and its potential role in multiple acyl-CoA dehydrogenase deficiency

Multiple acyl-CoA dehydrogenase deficiency (MADD) or glutaric aciduria type II (GAII) is most often caused by mutations in the genes encoding the α- or β-subunit of electron transfer flavoprotein (ETF) or electron transfer flavoprotein dehydrogenase (ETF-DH). Since not all patients have mutations in these genes, other as yet unidentified genes are predicted to be involved as well. Because all affected mitochondrial flavoproteins in MADD have FAD as a prosthetic group, the underlying defect in these patients may be due to a thus far undisclosed disturbance in the metabolism of FAD. Since a proper mitochondrial flavin balance is maintained by a mitochondrial FAD transporter, a defect of this transporter could also cause an MADD-like phenotype. In yeast, FAD is transported across the mitochondrial inner membrane by the FLX1 protein. An FLX1-mutated Saccharomyces cerevisiae strain exhibits a decreased activity of several mitochondrial flavoproteins. In the present study, we report the identification of the human mitochondrial FAD transporter. Based on sequence similarity to FLX1, we identified two human candidate genes (MFT and N111), which were cloned and characterized by functional expression in an FLX1-mutated yeast strain. Of the two candidate genes, only the previously described mitochondrial folate transporter (MFT) was able to functionally complement the FLX1 mutant. Candidates for mutations in the MFT gene are patients with a clinical suspicion of MADD but without any mutation in the α- or β-subunit of ETF or ETF-DH.

Differentially transcribed genes in skeletal muscle of Duroc and Taoyuan pigs1

The objective of this study was to compare gene transcription profiles of LM between two pig breeds, Duroc and Taoyuan, which display dramatically different postnatal muscle growth. We isolated LM from neonatal pigs, and the Duroc muscle length and mass were greater (P < 0.01) than for Taoyuan pigs; however, insignificant differences in the muscle fiber area and the percentage of fiber types were found. A human high-density complementary DNA (cDNA) microarray consisting of 9,182 probes was used to compare gene transcription profiles of LM between the two breeds. The results showed that the transcription level of 73 genes and 44 genes in Duroc LM were upregulated and down-regulated by at least 1.75-fold (P < 0.05) compared with Taoyuan, respectively. The strongly upregulated genes in Duroc pigs included those encoding the complex of myofibrillar proteins (e.g., myosin light and heavy chains, and troponin), ribosomal proteins, transcription regulatory proteins (e.g., skeletal muscle LIM protein 1 [SLIM1] and high-mobility group proteins), and energy metabolic enzymes (e.g., electron-transferring flavo-protein dehydrogenase, NADH dehydrogenase, malate dehydrogenase, and ATP synthases). The highly transcribed genes that encode energy metabolic enzymes indicate a more glycolytic metabolism in Duroc LM, thereby favoring carbohydrates rather than lipids for use as energy substrates in this tissue. The over-transcribed genes that encode skeletal muscle-predominant proteins or transcription regulators that control myogenesis and/or muscle growth suggest a general mechanism for the observed higher rate of postnatal muscle growth in Duroc pigs. The transcription of one such gene, SLIM1, was more highly transcribed (P < 0.01) in Duroc LM at birth and at postnatal d 7 than in Taoyuan. The transcription of SLIM1 increased (P < 0.05) in Duroc LM from neonate through 7 d of age, whereas its transcription remained essentially constant in Taoyuan during this period. These results suggest that SLIM1 may be useful for the development of markers associated with the postnatal muscle growth of pigs.

Assignment ofEtfdh, Etfb,andEtfato Chromosomes 3, 7, and 13: The Mouse Homologs of Genes Responsible for Glutaric Acidemia Type II in Human

Electron transfer flavoprotein (composed of α and β subunits) is an obligatory electron acceptor for several dehydrogenases and is located in the mitochondrial matrix. Electrons accepted by electron transfer flavoprotein (ETF) are transferred to the main mitochondrial respiratory chain by way of ETF dehydrogenase (ETFDH). In humans, deficiency of ETF or ETFDH leads to glutaric acidemia type II, an inherited metabolic disorder that can be fatal in its neonatal form and is characterized by severe hypoketotic hypoglycemia and acidosis. We used cDNA probes for theEtfdh, Etfb,andEtfagenes to determine localization of these mouse genes to chromosomes 3, 7, and 13.

Evidence for Intermediate Channeling in Mitochondrial β-Oxidation

The accumulation of beta-oxidation intermediates was studied by incubating normal and beta-oxidation enzyme-deficient human fibroblasts with [2H4]linoleate and L-carnitine and analyzing the resultant acylcarnitines by tandem mass spectrometry. Labeled decenoyl-, octanoyl-, hexanoyl-, and butyrylcarnitines were the only intermediates observed with normal cells. Intermediates of longer chain length, corresponding to substrates for the beta-oxidation enzymes associated with the inner mitochondrial membrane, were not observed unless a cell line was deficient in one of these enzymes, such as very-long-chain acyl-CoA dehydrogenase, long-chain 3-hydroxyacyl-CoA dehydrogenase, or electron transfer flavoprotein dehydrogenase. Matrix enzyme deficiencies, such as medium- and short-chain acyl-CoA dehydrogenases, were characterized by elevated concentrations of intermediates corresponding to their respective substrates (octanoyl- and decenoylcarnitines in medium-chain acyl-CoA dehydrogenase deficiency and butyrylcarnitine in short-chain acyl-CoA dehydrogenase deficiency). These observations agree with the notion of intermediate channeling due to the organization of beta-oxidation enzymes in complexes. The only exception is the incomplete channeling from thiolase to acyl-CoA dehydrogenase in the matrix. This situation may be a consequence of only one 3-ketoacyl-CoA thiolase being unable to interact with the several acyl-CoA dehydrogenases in the matrix.

Newly Identified Forms of Electron Transfer Flavoprotein Deficiency in Two Patients with Glutaric Aciduria Type II

Newly identified forms of electron transfer flavoprotein (ETF) deficiency in two patients with glutaric aciduria type II (GA II) were described. GA II has been attributed to a defect of either ETF or ETF dehydrogenase, resulting in multiple acyl-CoA dehydrogenation deficiency. ETF is a mitochondrial flavoprotein consisting of an alpha-subunit, alpha-ETF, and a beta-subunit, beta-ETF. We used pulse-chase experiments to examine the biosynthesis of ETF in fibroblasts from two patients with GA II. Patient 1 was a boy with the neonatal onset form, but without congenital anomalies, who is living at age 2 y. A defect of beta-ETF biosynthesis was noted in this patient. Patient 2 was a boy with the neonatal onset form with congenital anomalies who died on the 3rd postnatal day. He presented with a peculiar face and polycystic kidneys. In patient 2, both alpha- and beta-ETF were synthesized, but both the subunits were rapidly degraded. The lability of ETF was considered to be the cause of GA II in this patient. These two cases appear to be new forms of ETF deficiency in GA II.

7 Defects of fatty-acid oxidation in muscle

Long-chain fatty acids (LCFA) are oxidized by muscle mitochondria after transport in the cytosol by fatty-acid-binding protein(s) and their activation by a thiokinase. Carnitine, two forms of carnitine palmitoyltransferase(s) and carnitine acylcarnitine translocase are involved in LCFA gating. A primary genetic carnitine deficiency occurs in children with dilated cardiomyopathy, hypoglycaemia and low carnitine content in plasma, liver and muscle, owing to a defect in a common high-affinity transport system. This high-affinity transport in muscle differs from a low-affinity transport that has modifications during muscle maturation. The genetic enzyme defects of beta-oxidation (long-chain acyl-CoA dehydrogenase, medium- and short-chain acyl-CoA-dehydrogenase) present with Reye-like attacks that may lead to non-ketotic hypoglycaemia, coma and sudden infant death syndrome. There is elevated urinary excretion of dicarboxylic acids, acylcarnitines and acylglycines. Secondary carnitine deficiency may occur. ETF and ETF dehydrogenase deficiencies may present in a neonatal form with congenital anomalies, or in a later-onset form with ethylmalonic adipic aciduria. A still-unidentified defect leads to LCFA accumulation in fibroblasts, bone marrow, liver and muscle cells in a multisystem triglyceride disorder.

Glutaric Aciduria Type II: Autopsy Study of a Case with Electron-Transferring Flavoprotein Dehydrogenase Deficiency

An autopsy study of glutaric aciduria type II in a 62-day-old Japanese boy is presented. The diagnosis was made by analysis of organic acids in the urine. Immunoblot analysis of liver homogenate confirmed the diagnosis, revealing absence of electron-transferring flavoprotein dehydrogenase. The major findings were fatty changes of variable degree in many organs and tissues, the most severe being found in cardiac myocytes, hepatocytes, renal tubular epithelium, and skeletal muscle fibers. Other pertinent findings included multicystic and dysplastic kidney, pulmonary alveolar proteinosis, and spongiosis and gliosis of the spinal cord. The thymus was markedly depleted, and lymphocytes in the lymph nodes were mainly B cells. Although some of these changes may have been secondary to the sepsis and immunosuppression complicating 2 months of intensive care, the abnormal organic acid metabolism with severe acidosis may have been a significant contributing factor.

Acyl-CoA dehydrogenases, electron transfer flavoprotein and electron transfer flavoprotein dehydrogenase

Conference Article| June 01 1988 Acyl-CoA dehydrogenases, electron transfer flavoprotein and electron transfer flavoprotein dehydrogenase FRANK E. FRERMAN FRANK E. FRERMAN 1Department of Pediatrics, University of Colorado, School of Medicine, Denver, Colorado 80262, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1988) 16 (3): 416–418. https://doi.org/10.1042/bst0160416 Article history Received: November 22 1987 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share MailTo Twitter LinkedIn Cite Icon Cite Get Permissions Citation FRANK E. FRERMAN; Acyl-CoA dehydrogenases, electron transfer flavoprotein and electron transfer flavoprotein dehydrogenase. Biochem Soc Trans 1 June 1988; 16 (3): 416–418. doi: https://doi.org/10.1042/bst0160416 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: ETF, electron transferring flavoprotein, ETF—QO, electron transferring flavoprotein-ubiquinone oxido-reductase, GAD, general fatty acyl-CoA dehydrogenase, Ql… Qn, ubiquinone (n = 10) analogues This content is only available as a PDF. © 1988 Biochemical Society1988 Article PDF first page preview Close Modal You do not currently have access to this content.

831 MOLECULAR HETEROGENEITY OF ELECTRON TRANSFER FLAVOPROTEIN (ETF) IN GLUTARIC ACIDURIA TYPE II DUE TO AN ETF DEFICIENCY

Glutaric aciduria type II (GAII) is due to a deficiency of either ETF or ETF dehydrogenase. We studied the biosynthesis of ETF in 10 GAII lines as well as in normal controls. Cells were labeled with [35S]methionine in the presence and absence of rhodamine 6G. Rhodamine 6G inhibits mitochondrial ATP synthesis, thereby inhibiting post-translational processing of the nuclear coded mitochondrial enzymes. The labeled human ETF was immunoprecipitated from the solubilized cells using the anti-rat ETF antiserum and Staph A cells and analyzed by SDS-PAGE. In normal cells, α-subunit is synthesized as a precursor (35 kd), 3 kd larger than the mature counterpart (32 kd), while β-subunit is synthesized in a size indistinguishable from that of the mature β-subunit (27 kd). ETF abnormality was found in three GAII lines. In two of them, α-subunit was synthesized either not at all or in an extremely small amount. The size of the precursor and mature forms of ETF were of the normal sizes. In contrast, α-subunit synthesized in the third GAII line was 1 kd smaller than the normal size, both as precursor and mature form. In all other 7 GAII lines, the synthesis of α-subunit were normal. These 7 cell lines are due to an ETF dehydrogenase deficiency. Abnormality of β-subunit was found in none of the cell lines tested.

Riboflavin-responsive defects of beta-oxidation.

The key reaction in the beta-oxidation of fatty acids is the acyl-CoA dehydrogenation, catalyzed by short chain, medium chain, and long chain acyl-CoA dehydrogenases. Acyl-CoA dehydrogenation reactions are also involved in the metabolism of the branched chain amino acids, where isovaleryl-CoA and 2-methylbutyryl-CoA dehydrogenases are involved and in the metabolism of lysine, 5-hydroxylysine and tryptophan, where glutaryl-CoA dehydrogenase functions. In all of these dehydrogenation systems reducing equivalents are transported to the main respiratory chain by electron transfer flavoprotein (ETF) and electron transfer flavoprotein dehydrogenase (ETFDH), which are common to all the dehydrogenation systems. The acyl-CoA dehydrogenation enzymes are dependent on flavin adenine dinucleotide (FAD) as coenzyme, for which riboflavin is the precursor. Patients with multiple acyl-CoA dehydrogenation deficiencies have been found in whom the defect has been located to ETF and/or ETFDH. A few patients with multiple acyl-CoA dehydrogenation deficiencies have been described, in whom no defects in acyl-CoA dehydrogenases, ETF or ETFDH have been found but who respond clinically and biochemically to pharmacological doses of riboflavin. This indicates a defect related to the metabolism of FAD. An uptake defect of riboflavin or a synthesis defect of FAD from riboflavin have been excluded by in vivo and in vitro studies. A mitochondrial transport defect of FAD or a defect in the binding FAD to ETF and/or ETFDH remains possible.

Glutaric aciduria type II: evidence for a defect related to the electron transfer flavoprotein or its dehydrogenase.

Incubation of intact fibroblasts from a patients with glutaric aciduria type II with [2-14C]riboflavin showed normal synthesis of flavin mononucleotide and flavin adenine dinucleotide. This is taken as evidence for normal transport of riboflavin into the cells and normal activity of riboflavin kinase (EC 2.7.1.26) and flavin mononucleotide adenylyltransferase (EC 2.7.7.2). The ability of intact fibroblasts to oxidize 1-14C-fatty acids and [6-14C]lysine is impaired in the patient which together with the urinary excretion pattern of organic acids indicates a defective dehydrogenation of fatty acid acyl-CoAs and glutaryl-CoA. However, dehydrogenation of (C6-C10) fatty acid acyl-CoA derivatives and glutaryl-CoA was normal when the dehydrogenases were measured in fibroblast homogenate with artificial electron acceptors. In vivo, these dehydrogenases transfer their electrons to CoQ10 in the main electron transport chain via electron transfer flavoprotein and electron transfer flavoprotein dehydrogenase. Glutaric aciduria type II fibroblasts showed very diminished activity when the glutaryl-CoA dehydrogenase activity was measured without artificial electron acceptor but with intact endogenous electron transport system. As the NADH and succinate oxidation seems normal in glutaric aciduria type II patients, this is strong evidence for a defect in either the electron transfer flavoprotein or the electron transfer flavoprotein dehydrogenase.

ELECTRON-TRANSFERRING FLAVOPROTEIN (ETF) ACTIVITY IS DEFICIENT IN GLUTARIC ACIDURIA TYPE II (GA-II) AND ETHYLMALONIC-ADIPIC ACIDURIA (EMA) FIBROBLASTS

GA-II and a mild variant, EMA, result from impaired mitochondrial dehydrogenation of multiple acyl-CoA's, due to deficient ETF or ETF dehydrogenase (DH) activity, which transfer electrons from the acyl-CoA DH (ADH) to coenzyme Q. In two GA-II, one female and one male, and in 3 EMA/mild GA-II patients, medium chain ADH (MCADH) activities in GA-II and EMA fibroblast mitochondrial supernatants (MS), measured by a dye assay, were 55% (p<0.05) and 77% of control; with a 3H-release assay, MCADH activities in GA-II and EMA MS were 80% and 92% of control. In GA-II and EMA whole fibroblast supernatants, short chain ADH, MCADH, and isovaleryl-CoA DH activities ranged from 76% to 103% of control (p>0.1). With a dye reduction method in which MS ETF transfers electrons from 39 pmoles pure pig liver MCADH to an acceptor dye, ETF activity in GA-II was 29% (p<0.05; N=10) and in EMA 62% of control (p<0.05; N=14). When assayed with 165 pmoles MCADH, ETF activities in control MS increased three-fold and in GA-II and EMA MS were 8% (p<0.01) and 36% of control (p<0.05). Since flavin adenine dinucleotide (20μM) increased ETF activity in GA-II and EMA to only 15% and 51% of control, FAD binding to ETF is probably not defective in GA-II and EMA. Anti-mycin-A increased electron flux to the acceptor dye and control ETF activity 40%, but ETF activities in GA-II and EMA MS were still 15% and 46% of control. These cases of GA-II and EMA appear due to severe and mild ETF deficiencies, respectively.

Glutaryl-CoA dehydrogenase activity determined with intact electron-transport chain: application to glutaric aciduria type II.

Normal glutaric aciduria type II (GA II, McKusick 23168) is a fatal inborn error of metabolism with onset within the first days of life (Przyrembel et al., 1976; Gregersen et al., 1980). It is characterized by the urinary excretion of a number of organic acids that seem to be derived from substrates to a group of FAD containing acyl-CoA dehydrogenases. These dehydrogenases all transfer electrons to the electron-transport chain (ETC) through electron transfer flavoprotein (ETF) and ETF dehydrogenase (ETF DH).

Resonance Raman studies of ETF dehydrogenase (an iron sulfur flavoprotein)

ETF Dehydrogenase is an iron sulfur flavoprotein responsible for the transfer of electrons between electron transfer flavoprotein (ETF) and CoQ of the electron transport chain. We have determined the resonance Raman spectrum of this enzyme observing in the process at least seven of thirteen flavin bands in the 1100 cm−1–1600 cm−1 region of the Raman spectrum. The positions of three of these bands, II, IX, and X (see Figure I and Table I for band numbering system) in ETF dehydrogenase is very similar to their positions in aqueous solution of flavins in which water is hydrogen bonded to N-1, N-5, C=0(2), C=0(4), and N-H(3) of flavin. Conversely the positions of the flavin Raman bands are considerably shifted from those of flavin in nonhydrogen bonding solvent. The positions of bands II, IX, and X are nearly identical to those in the flavoprotein glutathione reductase; x-ray structural investigations on this enzyme indicate that there is extensive hydrogen bonding between FAD and protein in this molecule. A previous study in our laboratory has demonstrated that metal complexation at N-5 and C=0(4) with either Ru or Ag produces large shifts in the positions of Raman bands II, VI, IX, and X. None of these shifts are observed in ETF dehydrogenase indicating that there is no direct inner sphere coordination of Fe to flavin. In addition to the Raman bands of flavin observed in our spectrum, we also observe one band that is in the Fe-S stretching region observed for a variety of Fe-S proteins. This band is located at 331 cm−1. The frequency of the band corresponds to the 335 cm−1 band associated with the strongest Fe-S stretching mode in the 4Fe4S protein ferrodoxin from C. pasterianum. The observed frequency is quite different from that of the 3Fe3S proteins such as ferrodoxin(II) from D. gigas. Finally, ETF dehydrogenase shows no loss of activity or visual evidence of photodegradation in the laser beam as most other FeS proteins do

Effects of dietary iron deficiency on iron-sulfur proteins and bioenergetic functions of skeletal muscle mitochondria

Decreases in the mitochondrial content of Fe-S proteins appear to cause a loss of bioenergetic functions in skeletal muscle mitochondria during dietary iron deficiency. Following 21 days of an iron-deficient diet (2 mg iron/kg), significant decreases in muscle mitochondrial iron protein content, dehydrogenase and oxidase activities, and rates of ATP synthesis were observed relative to rats fed a control diet (50 mg iron/kg). Fe-S proteins were studied by EPR spectroscopy at low temperatures, following incubation with substrates, and/or redox titrations. Submitochondrial preparations were utilized in order to overcome membrane barriers to electron donors and to concentrate inner mitochondrial membrane Fe-S proteins. The Fe-S proteins of rat muscle mitochondria (not previously studied) appear remarkably similar to those of other tissues and species. Although the concentration of most Fe-S proteins studied was decreased by iron deficiency, the Rieske cluster of Complex III (Fe-S bc-1) was only minimally affected. In Complex II, Fe-S cluster S-1 was decreased 38% and cluster S-3 was decreased 59%. Complex I Fe-S clusters were substantially decreased by iron deficiency. The Fe-S cluster of electron-transferring flavoprotein dehydrogenase was 55% lower in the iron-deficient submitochondrial preparations. Succinate and NADH dehydrogenase activities, and maximal rates of reversed electron transport were about 80% lower in iron-deficient submitochondrial preparations than in controls, and oxidase activities were decreased about 70%. Decreases in cytochrome content were too small to explain loss of oxidase activity, and clearly could not affect dehydrogenases or reversed electron transport. Despite reductions in maximal rates of ATP formation, the efficiency of phosphorylative coupling was not affected. Our studies suggest that the loss of Fe-S clusters causes decreases in the dehydrogenase activities and that the iron depletion from the complexes is not a random process.

The pattern of iron—sulfur centers in brown adipose tissue mitochondria: Preponderance of ETF dehydrogenase and invariance with the thermogenic state

Most of the resonances observed in heart mitochondria by e.p.r. spectroscopy at presently attainable sensitivities have been recently identified (for review, see [ 1 ] ) inculding the so-called iron-sulfur (Fe-S) ‘center 5’ [2], recognized by its low field resonance (gz = 2.086) in the reduced state of mitochondria from a variety of species and tissues [l] . The assignment of this e.p.r. line was based on the purification of a thus far unknown iron-sulfur (Fe-S) flavoprotein from beef heart mitochondria [3]. Since the purified Fe-S flavoprotein was most readily reduced by fatty acyl-CoA in the presence of acyl-CoA dehydrogenase and electron-transferring flavoprotein (ETF), it was suggested [3] that this Fe-S flavoprotein may function as an electron transfer protein between the fatty acyl-CoA dehydrogenase system [4] and the terminal part of the respiratory chain. In support of this conclusion, we would like to present further experimental evidence based on e.p.r. spectra of mitochondria from brown adipose tissue, a tissue highly specialized for fatty acid oxidation and non-shivering thermogenesis (for review, see [5]). Furthermore, we want to present results of e.p.r. measurements on whole brown adipose tissue during the entire period of adaptation to a cold environment, which establishes the suitability of e.p.r. spectroscopy as a non-