Electron-transferring flavoprotein and its dehydrogenase contributed to growth development and virulence in Beauveria bassiana.

Electron-transferring flavoprotein (Etf) and its dehydrogenase (Etfdh) are integral components of the electron transport chain in mitochondria. In this study, we characterize two putative etf genes (Bbetfa and Bbetfb) and their dehydrogenase gene Bbetfdh in the entomopathogenic fungus Beauveria bassiana. Individual deletion of these genes caused a significant reduction in vegetative growth, conidiation, and delayed conidial germination. Lack of these genes also led to abnormal metabolism of fatty acid and increasing lipid body accumulation. Furthermore, the virulence of Bbetfs and Bbetfdh deletion mutants was severely impaired due to decreasing infection structure formation. Additionally, all deletion strains showed reduced ATP synthesis compared to the wild-type strain. Taken together, Bbetfa and Bbetfb, along with Bbetfdh, play principal roles in fungal vegetative growth, conidiation, conidial germination, and pathogenicity of B. bassiana due to their essential functions in fatty acid metabolism.

Rapid-reaction kinetics of the butyryl-CoA dehydrogenase component of the electron-bifurcating crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from Megasphaera elsdenii.

We have investigated the equilibrium properties and rapid-reaction kinetics of the isolated butyryl-CoA dehydrogenase (bcd) component of the electron-bifurcating crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase (EtfAB-bcd) from Megasphaera elsdenii. We find that a neutral FADH• semiquinone accumulates transiently during both reduction with sodium dithionite and with NADH in the presence of catalytic concentrations of EtfAB. In both cases full reduction of bcd to the hydroquinone is eventually observed, but the accumulation of FADH• indicates that a substantial portion of reduction occurs in sequential one-electron processes rather than a single two-electron event. In rapid-reaction experiments following the reaction of reduced bcd with crotonyl-CoA and oxidized bcd with butyryl-CoA, long-wavelength-absorbing intermediates are observed that are assigned to bcdred:crotonyl-CoA and bcdox:butyryl-CoA charge-transfer complexes, demonstrating their kinetic competence in the course of the reaction. In the presence of crotonyl-CoA there is an accumulation of semiquinone that is unequivocally the anionic FAD•- rather than the neutral FADH• seen in the absence of substrate, indicating that binding of substrate/product results in ionization of the bcd semiquinone. In addition to fully characterizing the rapid-reaction kinetics of both the oxidative and reductive half-reactions, our results demonstrate that one-electron processes play an important role in the reduction of bcd in EtfAB-bcd.

Functional and structural impact of 10 ACADM missense mutations on human medium chain acyl-Coa dehydrogenase.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency (MCADD) is associated with ACADM gene mutations, leading to an impaired function and/or structure of MCAD. Importantly, after import into the mitochondria, MCAD must incorporate a molecule of flavin adenine dinucleotide (FAD) per subunit and assemble into tetramers. However, the effect of MCAD amino acid substitutions on FAD incorporation has not been investigated. Herein, the commonest MCAD variant (p.K304E) and 11 additional rare variants (p.Y48C, p.R55G, p.A88P, p.Y133C, p.A140T, p.D143V, p.G224R, p.L238F, p.V264I, p.Y372N, and p.G377V) were functionally and structurally characterized. Half of the studied variants presented a FAD content <65 % compared to the wild-type. Most of them were recovered as tetramers, except the p.Y372N (mainly as dimers). No correlation was found between the levels of tetramers and FAD content. However, a correlation between FAD content and the cofactor's affinity, proteolytic stability, thermostability, and thermal inactivation was established. We showed that the studied amino acid changes in MCAD may alter the substrate chain-length dependence and the interaction with electron-transferring-flavoprotein (ETF) necessary for a proper functioning electron transfer thus adding additional layers of complexity to the pathological effect of ACADM missense mutations. Although the majority of the variant MCADs presented an impaired capacity to retain FAD during their synthesis, some of them were structurally rescued by cofactor supplementation, suggesting that in the mitochondrial environment the levels and activity of those variants may be dependent of FAD's availability thus contributing for the heterogeneity of the MCADD phenotype found in patients presenting the same genotype.

Unusual reactivity of a flavin in a bifurcating electron-transferring flavoprotein leads to flavin modification and a charge-transfer complex.

From the outset, canonical electron transferring flavoproteins (ETFs) earned a reputation for containing modified flavin. We now show that modification occurs in the recently recognized bifurcating (Bf) ETFs as well. In Bf ETFs, the 'electron transfer' (ET) flavin mediates single electron transfer via a stable anionic semiquinone state, akin to the FAD of canonical ETFs, whereas a second flavin mediates bifurcation (the Bf FAD). We demonstrate that the ET FAD undergoes transformation to two different modified flavins by a sequence of protein-catalyzed reactions that occurs specifically in the ET site, when the enzyme is maintained at pH 9 in an amine-based buffer. Our optical and mass spectrometric characterizations identify 8-formyl flavin early in the process and 8-amino flavins (8AFs) at later times. The latter have not previously been documented in an ETF to our knowledge. Mass spectrometry of flavin products formed in Tris or bis-tris-aminopropane solutions demonstrates that the source of the amine adduct is the buffer. Stepwise reduction of the 8AF demonstrates that it can explain a charge transfer band observed near 726 nm in Bf ETF, as a complex involving the hydroquinone state of the 8AF in the ET site with the oxidized state of unmodified flavin in the Bf site. This supports the possibility that Bf ETF can populate a conformation enabling direct electron transfer between its two flavins, as has been proposed for cofactors brought together in complexes between ETF and its partner proteins.

Structure-based electron-confurcation mechanism of the Ldh-EtfAB complex.

Lactate oxidation with NAD+ as electron acceptor is a highly endergonic reaction. Some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) using a lactate dehydrogenase (Ldh) in concert with the electron-transferring proteins EtfA and EtfB. The electron cryo-microscopically characterized (Ldh-EtfAB)2 complex of Acetobacterium woodii at 2.43 Å resolution consists of a mobile EtfAB shuttle domain located between the rigid central Ldh and the peripheral EtfAB base units. The FADs of Ldh and the EtfAB shuttle domain contact each other thereby forming the D (dehydrogenation-connected) state. The intermediary Glu37 and Glu139 may harmonize the redox potentials between the FADs and the pyruvate/lactate pair crucial for FBEC. By integrating Alphafold2 calculations a plausible novel B (bifurcation-connected) state was obtained allowing electron transfer between the EtfAB base and shuttle FADs. Kinetic analysis of enzyme variants suggests a correlation between NAD+ binding site and D-to-B-state transition implicating a 75° rotation of the EtfAB shuttle domain. The FBEC inactivity when truncating the ferredoxin domain of EtfA substantiates its role as redox relay. Lactate oxidation in Ldh is assisted by the catalytic base His423 and a metal center. On this basis, a comprehensive catalytic mechanism of the FBEC process was proposed.

Exploring Thermal Sensitivities and Adaptations of Oxidative Phosphorylation Pathways.

Temperature shifts are a major challenge to animals; they drive adaptations in organisms and species, and affect all physiological functions in ectothermic organisms. Understanding the origin and mechanisms of these adaptations is critical for determining whether ectothermic organisms will be able to survive when faced with global climate change. Mitochondrial oxidative phosphorylation is thought to be an important metabolic player in this regard, since the capacity of the mitochondria to produce energy greatly varies according to temperature. However, organism survival and fitness depend not only on how much energy is produced, but, more precisely, on how oxidative phosphorylation is affected and which step of the process dictates thermal sensitivity. These questions need to be addressed from a new perspective involving a complex view of mitochondrial oxidative phosphorylation and its related pathways. In this review, we examine the effect of temperature on the commonly measured pathways, but mainly focus on the potential impact of lesser-studied pathways and related steps, including the electron-transferring flavoprotein pathway, glycerophosphate dehydrogenase, dihydroorotate dehydrogenase, choline dehydrogenase, proline dehydrogenase, and sulfide:quinone oxidoreductase. Our objective is to reveal new avenues of research that can address the impact of temperature on oxidative phosphorylation in all its complexity to better portray the limitations and the potential adaptations of aerobic metabolism.

The reductive half-reaction of two bifurcating electron-transferring flavoproteins: Evidence for changes in flavin reduction potentials mediated by specific conformational changes.

The EtfAB components of two bifurcating flavoprotein systems, the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from the bacterium Megasphaera elsdenii and the menaquinone-dependent NADH:ferredoxin oxidoreductase from the archaeon Pyrobaculum aerophilum, have been investigated. With both proteins, we find that removal of the electron-transferring flavin adenine dinucleotide (FAD) moiety from both proteins results in an uncrossing of the reduction potentials of the remaining bifurcating FAD; this significantly stabilizes the otherwise very unstable semiquinone state, which accumulates over the course of reductive titrations with sodium dithionite. Furthermore, reduction of both EtfABs depleted of their electron-transferring FAD by NADH was monophasic with a hyperbolic dependence of reaction rate on the concentration of NADH. On the other hand, NADH reduction of the replete proteins containing the electron-transferring FAD was multiphasic, consisting of a fast phase comparable to that seen with the depleted proteins followed by an intermediate phase that involves significant accumulation of FAD⋅-, again reflecting uncrossing of the half-potentials of the bifurcating FAD. This is then followed by a slow phase that represents the slow reduction of the electron-transferring FAD to FADH-, with reduction of the now fully reoxidized bifurcating FAD by a second equivalent of NADH. We suggest that the crossing and uncrossing of the reduction half-potentials of the bifurcating FAD is due to specific conformational changes that have been structurally characterized.

Screening of multiple acyl-CoA dehydrogenase deficiency in newborns and follow-up of patients.

To investigate the incidence rate, clinical and gene mutation characteristics of multiple acyl-CoA dehydrogenase deficiency (MADD) in newborns in Zhejiang province. A total of 3 896 789 newborns were screened for MADD using tandem mass spectrometry in Zhejiang Neonatal Screening Center during January 2009 and December 2020. Patients of MADD were confirmed by urine organic acid and electron transferring flavoprotein (or electron transferring flavoprotein dehydrogenase () gene detection. MADD patients were given diet and life management, supplemented with L-carnitine, riboflavin and coenzyme Q 10 treatment, and their growth and intellectual development were evaluated during the followed up.Thirteen patients with MADD were diagnosed, with an incidence of 1/299 753. One patient was type Ⅱ, and the rest were type Ⅲ. Patients were followed up for 1 case died, 4 cases had acute metabolic disorders with hypoglycemia as the main manifestation due to infection, 1 case had hypotonia, and the rest 7 cases developed well. Patients had raised levels of C4-C18:1 acylcarnitines in the initial screening. Thirteen children were genetically tested, 1 case with compound heterozygous mutation in the gene, 1 case with homozygous mutation in the gene, 1 case with compound heterozygous mutation in the gene, 8 cases with compound heterozygous mutation and 1 case with homozygous mutation in the gene, 1 case that only 1 locus of gene was detected. The c.250G>A was the hotspot mutation in this study.The clinical manifestations of MADD are highly heterogeneous. The neonatal-onset form is serious, and late onset form usually has no obvious clinical symptoms. C4-C18:1 acylcarnitines usually increased in the initial screening, and the hotspot gene mutation is c.250G>A.

Biochemical characterization of human D-2-hydroxyglutarate dehydrogenase and two disease related variants reveals the molecular cause of D-2-hydroxyglutaric aciduria.

D-2-hydroxyglutaric aciduria is a neurometabolic disorder, characterized by the accumulation of D-2-hydroxyglutarate (D-2HG) in human mitochondria. Increased levels of D-2HG are detected in humans exhibiting point mutations in the genes encoding isocitrate dehydrogenase, citrate carrier, the electron transferring flavoprotein (ETF) and its downstream electron acceptor ETF-ubiquinone oxidoreductase or D-2-hydroxyglutarate dehydrogenase (hD2HGDH). However, while the pathogenicity of several amino acid replacements in the former four proteins has been studied extensively, not much is known about the effect of certain point mutations on the biochemical properties of hD2HGDH. Therefore, we recombinantly produced wild type hD2HGDH as well as two recently identified disease-related variants (hD2HGDH-I147S and -V444A) and performed their detailed biochemical characterization. We could show that hD2HGDH is a FAD dependent protein, which is able to catalyze the oxidation of D-2HG and D-lactate to α-ketoglutarate and pyruvate, respectively. The two variants were obtained as apo-proteins and were thus catalytically inactive. The addition of FAD failed to restore enzymatic activity of the variants, indicating that the cofactor binding site is compromised by the single amino acid replacements. Further analyses revealed that both variants form aggregates that are apparently unable to bind the FAD cofactor. Since, D-2-hydroxyglutaric aciduria may also result from a loss of function of either the ETF or its downstream electron acceptor ETF-ubiquinone oxidoreductase, ETF may serve as the cognate electron acceptor of reduced hD2HGDH. Here, we show that hD2HGDH directly reduces recombinant human ETF, thus establishing a metabolic link between the oxidation of D-2-hydroxyglutarate and the mitochondrial electron transport chain.

Closing the gap: yeast electron-transferring flavoprotein links the oxidation of d-lactate and d-α-hydroxyglutarate to energy production via the respiratory chain.

Electron-transferring flavoproteins (ETFs) have been found in all kingdoms of life, mostly assisting in shuttling electrons to the respiratory chain for ATP production. While the human (h) ETF has been studied in great detail, very little is known about the biochemical properties of the homologous protein in the model organism Saccharomyces cerevisiae (yETF). In view of the absence of client dehydrogenases, for example, the acyl-CoA dehydrogenases involved in the ß-oxidation of fatty acids, d-lactate dehydrogenase 2 (Dld2) appeared to be the only relevant enzyme that is serviced by yETF for electron transfer to the mitochondrial electron transport chain. However, this hypothesis was never tested experimentally. Here, we report the biochemical properties of yETF and Dld2 as well as the electron transfer reaction between the two proteins. Our study revealed that Dld2 oxidizes d-α-hydroxyglutarate more efficiently than d-lactate exhibiting kcatapp /KMapp values of 1200 ± 300 m-1 ·s-1 and 11 ± 2 m-1 ·s-1 , respectively. As expected, substrate-reduced Dld2 very slowly reacted with oxygen or the artificial electron acceptor 2,6-dichlorophenol indophenol. However, photoreduced Dld2 was rapidly reoxidized by oxygen, suggesting that the reaction products, that is, α-ketoglutarate and pyruvate, 'lock' the reduced enzyme in an unreactive state. Interestingly, however, we could demonstrate that substrate-reduced Dld2 rapidly transfers electrons to yETF. Therefore, we conclude that the formation of a product-reduced Dld2 complex suppresses electron transfer to dioxygen but favors the rapid reduction in yETF, thus preventing the loss of electrons and the generation of reactive oxygen species.

Molecular basis of the flavin-based electron-bifurcating caffeyl-CoA reductase reaction.

Flavin-based electron bifurcation (FBEB) is a recently discovered mode of energy coupling in anaerobic microorganisms. The electron-bifurcating caffeyl-CoA reductase (CarCDE) catalyzes the reduction of caffeyl-CoA and ferredoxin by oxidizing NADH. The 3.5 Å structure of the heterododecameric Car(CDE)4 complex of Acetobacterium woodii, presented here, reveals compared to other electron-transferring flavoprotein/acyl dehydrogenase family members an additional ferredoxin-like domain with two [4Fe-4S] clusters N-terminally fused to CarE. It might serve, in vivo, as specific adaptor for the physiological electron acceptor. Kinetic analysis of a CarCDE(∆Fd) complex indicates the bypassing of the ferredoxin-like domain by artificial electron acceptors. Site-directed mutagenesis studies substantiated the crucial role of the C-terminal arm of CarD and of ArgE203, hydrogen-bonded to the bifurcating FAD, for FBEB.

Defining Electron Bifurcation in the Electron-Transferring Flavoprotein Family.

Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes.IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs.

Reduction of ferredoxin or oxygen by flavin-based electron bifurcation in Megasphaera elsdenii.

Over 50 years ago, it was reported that, in the anaerobic rumen bacterium Megasphaera elsdenii, the reduction of crotonyl-CoA to butyryl-CoA by NADH involved an electron transferring flavoprotein (Etf) as mediator [Baldwin RL, Milligan LP (1964) Biochim Biophys Acta 92, 421-432]. Purification and spectroscopic characterization revealed that this Etf contained 2 FAD, whereas, in the Etfs from aerobic and facultative bacteria, one FAD is replaced by AMP. Recently we detected a similar system in the related anaerobe Acidaminococcus fermentans that differed in the requirement of additional ferredoxin as electron acceptor. The whole process was established as flavin-based electron bifurcation in which the exergonic reduction of crotonyl-CoA by NADH mediated by Etf + butyryl-CoA dehydrogenase (Bcd) was coupled to the endergonic reduction of ferredoxin also by NADH. In the present study, we demonstrate that, under anaerobic conditions, Etf + Bcd from M. elsdenii bifurcate as efficiently as Etf + Bcd from A. fermentans. Under the aerobic conditions used in the study by Baldwin and Milligan and in the presence of catalytic amounts of crotonyl-CoA or butyryl-CoA, however, Etf + Bcd act as NADH oxidase producing superoxide and H2 O2 , whereas ferredoxin is not required. We hypothesize that, during bifurcation, oxygen replaces ferredoxin to yield superoxide. In addition, the formed butyryl-CoA is re-oxidized by a second oxygen molecule to crotonyl-CoA, resulting in a stoichiometry of 2 NADH consumed and 2 H2 O2 formed. As a result of the production of reactive oxygen species, electron bifurcation can be regarded as an Achilles' heel of anaerobes when exposed to air.

Kinetic and spectral properties of isovaleryl-CoA dehydrogenase and interaction with ligands.

Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and the transfer of electrons to the electron transfer flavoprotein (ETF). Recombinant human IVD purifies with bound CoA-persulfide. A modified purification protocol was developed to isolate IVD without bound CoA-persulfide and to protect the protein thiols from oxidation. The CoA-persulfide-free IVD specific activity was 112.5 µmol porcine ETF min(-)(1) mg(-)(1), which was ∼20-fold higher than that of its CoA-persulfide bound form. The Km and catalytic efficiency (kcat/Km) for isovaleryl-CoA were 1.0 µM and 4.3 × 10(6) M(-1) s(-1) per monomer, respectively, and its Km for ETF was 2.0 µM. Anaerobic titration of isovaleryl-CoA into an IVD solution resulted in a stable blue complex with increased absorbance at 310 nm, decreased absorbance at 373 and 447 nm, and the appearance of the charge transfer complex band at 584 nm. The apparent dissociation constant (KDapp) determined spectrally for isovaleryl-CoA was 0.54 µM. Isovaleryl-CoA, acetoacetyl-CoA, methylenecyclopropyl-acetyl-CoA, and ETF induced CD spectral changes at the 250-500 nm region while isobutyryl-CoA did not, suggesting conformational changes occur at the flavin ring that are ligand specific. Replacement of the IVD Trp166 with a Phe did not block IVD interaction with ETF, indicating that its indole ring is not essential for electron transfer to ETF. A twelve amino acid synthetic peptide that matches the sequence of the ETF docking peptide competitively inhibited the enzyme reaction when ETF was used as the electron acceptor with a Ki of 1.5 mM.

Glutaryl-coenzyme A dehydrogenase from Geobacter metallireducens - interaction with electron transferring flavoprotein and kinetic basis of unidirectional catalysis.

Glutaryl-CoA dehydrogenases (GDHs) are FAD containing acyl-CoA dehydrogenases that usually catalyze the dehydrogenation and decarboxylation of glutaryl-CoA to crotonyl-CoA with an electron transferring flavoprotein (ETF) acting as natural electron acceptor. In anaerobic bacteria, GDHs play an important role in the benzoyl-CoA degradation pathway of monocyclic aromatic compounds. In the present study, we identified, purified and characterized the benzoate-induced BamOP as the electron accepting ETF of GDH (BamM) from the Fe(III)-respiring Geobacter metallireducens. The BamOP heterodimer contained FAD and AMP as cofactors. In the absence of an artificial electron acceptor, at pH values above 8, the BamMOP-components catalyzed the expected glutaryl-CoA oxidation to crotonyl-CoA and CO2 ; however, at pH values below 7, the redox-neutral glutaryl-CoA conversion to butyryl-CoA and CO2 became the dominant reaction. This previously unknown, strictly ETF-dependent coupled glutaryl-CoA oxidation/crotonyl-CoA reduction activity was facilitated by an unexpected two-electron transfer between FAD(BamM) and FAD(BamOP) , as well as by the similar redox potentials of the two FAD cofactors in the substrate-bound state. The strict order of electron/proton transfer and C-C-cleavage events including transient charge-transfer complexes did not allow an energetic coupling of electron transfer and decarboxylation. This explains why it was difficult to release the glutaconyl-CoA intermediate from reduced GDH. Moreover, it provides a kinetic rational for the apparent inability of BamM to catalyze the reverse reductive crotonyl-CoA carboxylation, even under thermodynamically favourable conditions. For this reason reductive crotonyl-CoA carboxylation, a key reaction in C2-assimilation via the ethylmalonyl-CoA pathway, is accomplished by a different crotonyl-CoA carboxylase/reductase via a covalent NADPH/ene-adduct.

Regulation of energy metabolism by long-chain fatty acids.

In mammals, excess energy is stored primarily as triglycerides, which are mobilized when energy demands arise. This review mainly focuses on the role of long chain fatty acids (LCFAs) in regulating energy metabolism as ligands of peroxisome proliferator-activated receptors (PPARs). PPAR-alpha expressed primarily in liver is essential for metabolic adaptation to starvation by inducing genes for beta-oxidation and ketogenesis and by downregulating energy expenditure through fibroblast growth factor 21. PPAR-delta is highly expressed in skeletal muscle and induces genes for LCFA oxidation during fasting and endurance exercise. PPAR-delta also regulates glucose metabolism and mitochondrial biogenesis by inducing FOXO1 and PGC1-alpha. Genes targeted by PPAR-gamma in adipocytes suggest that PPAR-gamma senses incoming non-esterified LCFAs and induces the pathways to store LCFAs as triglycerides. Adiponectin, another important target of PPAR-gamma may act as a spacer between adipocytes to maintain their metabolic activity and insulin sensitivity. Another topic of this review is effects of skin LCFAs on energy metabolism. Specific LCFAs are required for the synthesis of skin lipids, which are essential for water barrier and thermal insulation functions of the skin. Disturbance of skin lipid metabolism often causes apparent resistance to developing obesity at the expense of normal skin function.

Sites of reactive oxygen species generation by mitochondria oxidizing different substrates.

Mitochondrial radical production is important in redox signaling, aging and disease, but the relative contributions of different production sites are poorly understood. We analyzed the rates of superoxide/H2O2 production from different defined sites in rat skeletal muscle mitochondria oxidizing a variety of conventional substrates in the absence of added inhibitors: succinate; glycerol 3-phosphate; palmitoylcarnitine plus carnitine; or glutamate plus malate. In all cases, the sum of the estimated rates accounted fully for the measured overall rates. There were two striking results. First, the overall rates differed by an order of magnitude between substrates. Second, the relative contribution of each site was very different with different substrates. During succinate oxidation, most of the superoxide production was from the site of quinone reduction in complex I (site IQ), with small contributions from the flavin site in complex I (site IF) and the quinol oxidation site in complex III (site IIIQo). However, with glutamate plus malate as substrate, site IQ made little or no contribution, and production was shared between site IF, site IIIQo and 2-oxoglutarate dehydrogenase. With palmitoylcarnitine as substrate, the flavin site in complex II (site IIF) was a major contributor (together with sites IF and IIIQo), and with glycerol 3-phosphate as substrate, five different sites all contributed, including glycerol 3-phosphate dehydrogenase. Thus, the relative and absolute contributions of specific sites to the production of reactive oxygen species in isolated mitochondria depend very strongly on the substrates being oxidized, and the same is likely true in cells and in vivo.

Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii.

Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii is a heterodimer containing two FAD cofactors. Isolated ETF contains only one FAD molecule, FAD-1, because the other, FAD-2, is lost during purification. FAD-2 is recovered by adding FAD to the isolated ETF. The two FAD molecules in holoETF were characterized using NADH. Spectrophotometric titration of isolated ETF with NADH showed a two-electron reduction of FAD-1 according to a monophasic profile indicating that FAD-1 receives electrons from NADH without involvement of FAD-2. When holoETF was titrated with NADH, FAD-2 was reduced to an anionic semiquinone and then was fully reduced before the reduction of FAD-1. The midpoint potential values at pH 7 were +81, -136 and -279 mV for the reduction of oxidized FAD-2 to semiquinone, semiquinone to the fully reduced FAD-2 and the two-electron reduction of FAD-1, respectively. Both FAD-1 and FAD-2 in holoETF were reduced by excess NADH very rapidly. The reduction of FAD-2 was slowed by replacement of FAD-1 with 8-cyano-FAD indicating that FAD-2 receives electrons from FAD-1 but not from NADH directly. The present results suggest that FAD-2 is the counterpart of the FAD in human ETF, which contains one FAD and one AMP.

Decomposition of the fluorescence spectra of two FAD molecules in electron-transferring flavoprotein from Megasphaera elsdenii.

Electron-transferring flavoprotein (ETF) from Megasphaera elsdenii contains two FAD molecules, FAD-1 and FAD-2. FAD-2 shows an unusual absorption spectrum with a 400-nm peak. In contrast, ETFs from other sources such as pig contain one FAD and one AMP with the FAD showing a typical flavin absorption spectrum with 380- and 440-nm peaks. It is presumed that FAD-2 is the counterpart of the FAD in other ETFs. In this study, the FAD-1 and FAD-2 fluorescence spectra were determined by titration of FAD-1-bound ETF with FAD using excitation-emission matrix (EEM) fluorescence spectroscopy. The EEM data were globally analysed, and the FAD fluorescence spectra were calculated from the principal components using their respective absorption spectra. The FAD-2 fluorescence spectrum was different from that of pig ETF, which is more intense and blue-shifted. AMP-free pig ETF in acidic solution, which has a comparable absorption spectrum to FAD-2, also had a similar fluorescence spectrum. This result suggests that FAD-2 in M. elsdenii ETF and the FAD in acidic AMP-free pig ETF share a common microenvironment. A review of published ETF fluorescence spectra led to the speculation that the majority of ETF molecules in solution are in the conformation depicted by the crystal structure.

Sites of superoxide and hydrogen peroxide production during fatty acid oxidation in rat skeletal muscle mitochondria.

H2O2 production by skeletal muscle mitochondria oxidizing palmitoylcarnitine was examined under two conditions: the absence of respiratory chain inhibitors and the presence of myxothiazol to inhibit complex III. Without inhibitors, respiration and H2O2 production were low unless carnitine or malate was added to limit acetyl-CoA accumulation. With palmitoylcarnitine alone, H2O2 production was dominated by complex II (44% from site IIF in the forward reaction); the remainder was mostly from complex I (34%, superoxide from site IF). With added carnitine, H2O2 production was about equally shared between complexes I, II, and III. With added malate, it was 75% from complex III (superoxide from site IIIQo) and 25% from site IF. Thus complex II (site IIF in the forward reaction) is a major source of H2O2 production during oxidation of palmitoylcarnitine ± carnitine. Under the second condition (myxothiazol present to keep ubiquinone reduced), the rates of H2O2 production were highest in the presence of palmitoylcarnitine ± carnitine and were dominated by complex II (site IIF in the reverse reaction). About half the rest was from site IF, but a significant portion, ∼40pmol H2O2·min(-1)·mg protein(-1), was not from complex I, II, or III and was attributed to the proteins of ß-oxidation (electron-transferring flavoprotein (ETF) and ETF-ubiquinone oxidoreductase). The maximum rate from the ETF system was ∼200pmol H2O2·min(-1)·mg protein(-1) under conditions of compromised antioxidant defense and reduced ubiquinone pool. Thus complex II and the ETF system both contribute to H2O2 productionduring fatty acid oxidation under appropriate conditions.