Electron Transfer Proteins Research Articles

Termination of Biological Function at Low Temperatures: Glass or Structural Transition?

Energy of life is produced by electron transfer in energy chains of respiration or photosynthesis. A small input of free energy available to biology puts significant restrictions on how much free energy can be lost in each electron-transfer reaction. We advocate the view that breaking ergodicity, leading to violation of the fluctuation-dissipation theorem (FDT), is how proteins achieve high reaction rates without sacrificing the reaction free energy. Here we show that a significant level of nonergodicity, represented by a large extent of the configurational temperature over the kinetic temperature, is maintained in the entire physiological range for the cytochrome c electron transfer protein. The protein returns to the state consistent with the FDT below the crossover temperature close to the temperature of the protein glass transition. This crossover leads to a sharp increase in the activation barrier of electron transfer and is displayed by a kink in the Arrhenius plot for the reaction rate constant.

Enhanced direct electron transfer of redox protein based on multiporous SnO2 nanofiber-carbon nanotube nanocomposite and its application in biosensing

A novel third generation H2O2 biosensor is fabricated using multiporous SnO2 nanofiber/carbon nanotubes (CNTs) composite as a matrix for the immobilization of redox protein onto glassy carbon electrode. The multiporous nanofiber (MPNFs) of SnO2 is synthesized by electrospinning technique from the tin precursor. This nanofiber shows high surface area and good electrical conductivity. The SnO2 nanofiber/CNT composite increases the efficiency of biomolecule loading due to its high surface area. The morphology of the nanofiber has been evaluated by scanning electron microscopy (SEM). Cyclic Voltammetry and amperometry technique are employed to study and optimize the performance of the fabricated electrode. A direct electron transfer between the protein's redox centre and the glassy carbon electrode is established after fabrication of the electrode. The fabricated electrode shows excellent electrocatalytic reduction to H2O2. The catalysis currents increases linearly to the H2O2 concentration in a wide range of 1.0 10−6–1.4×10−4M and the lowest detection limit was 30nM (S/N=3). Moreover, the biosensor showed a rapid response to H2O2, a good stability and reproducibility.

Patient with multiple acyl-CoA dehydrogenase deficiency disease and ETFDH mutations benefits from riboflavin therapy: a case report

BackgroundLipid storage myopathy (LSM) is a diverse group of lipid metabolic disorders with great variations in the clinical phenotype and age of onset. Classical multiple acyl-CoA dehydrogenase deficiency (MADD) is known to occur secondary to mutations in electron transfer flavoprotein dehydrogenase (ETFDH) gene. Whole exome sequencing (WES) with clinical correlations can be useful in identifying genomic alterations for targeted therapy.Case presentationWe report a patient presented with severe muscle weakness and exercise intolerance, suggestive of LSM. Diagnostic testing demonstrated lipid accumulation in muscle fibres and elevated plasma acyl carnitine levels. Exome sequencing of the proband and two of his unaffected siblings revealed compound heterozygous mutations, c.250G > A (p.Ala84Thr) and c.770A > G (p.Tyr257Cys) in the ETFDH gene as the probable causative mutations. In addition, a previously unreported variant c.1042C > T (p.Arg348Trp) in ACOT11 gene was found. This missense variant was predicted to be deleterious but its association with lipid storage in muscle is unclear. The diagnosis of MADD was established and the patient was treated with riboflavin which resulted in rapid clinical and biochemical improvement.ConclusionsOur findings support the role of WES as an effective tool in the diagnosis of highly heterogeneous disease and this has important implications in the therapeutic strategy of LSM treatment.

Photoinduced electron-transfer reactions of tris(2,2′-bipyridine)ruthenium(II)-based carbonic anhydrase inhibitors tethering plural binding sites

AbstractTris(2,2′‐bipyridine)ruthenium(II) complex‐based carbonic anhydrase (CA) inhibitors, [Ru(bpy)2(bpydbs)]2+ {bpy = 2,2′‐bipyridine and bpydbs = 2,2′‐bipyridinyl‐4,4′‐dicarboxilic acid bis[(2‐{2‐[2‐(4‐sulfamoylbenzoylamino)ethoxy]ethoxy}ethyl)amide]} and [Ru(bpydbs)3]2+, tethering plural benzenesulfonamide groups have been prepared. The CA catalytic activity was effectively suppressed by these synthetic [Ru(bpy)2(bpydbs)]2+ and [Ru(bpydbs)3]2+ inhibitors, and their dissociation constants at pH = 7.2 and at 25°C were determined to be KI = 0.93 ± 0.02 μM and KI = 0.24 ± 0.03 μM, respectively. Next, 2 photoinduced electron‐transfer (ET) systems comprising a Ru2+‐CA complex and an electron acceptor, such as chloropentaamminecobalt(III) ([CoCl(NH3)5]2+) or methylviologen (MV2+) were studied. In the presence of CA and a sacrificial electron acceptor, such as pentaamminechlorocobalt(III) complex, the photoexcited triplet state of 3([Ru(II)]2+)* was quenched through an intermolecular photoinduced ET mechanism. In case of the [Ru(bpydbs)3]2+‐CA‐MV2+ system, the photoexcited triplet state of 3([Ru(bpydbs)3]2+)* was quenched by sacrificial quencher through an intermolecular photoinduced ET mechanism, giving the oxidized [Ru(bpydbs)3]3+. Then the following intramolecular ET from the amino acid residue, Tyr6, near the active site of CA proceeded. We observed a transient absorption around at 410 nm, arising from the formation of a Tyr•+ in the [Ru(bpydbs)3]2+‐CA‐MV2+ system. These artificial Ru(II)‐CA systems may clearly demonstrate both intermolecular and intramolecular photoinduced ET reactions of protein and could be one of the interesting models of the ET proteins. Their photophysical properties and the detailed ET mechanisms are discussed in order to clarify the multistep ET reactions.

Absolute quantification of selected photosynthetic electron transfer proteins in Chlamydomonas reinhardtii in the presence and absence of oxygen.

The absolute amount of plastocyanin (PC), ferredoxin-NADP+-oxidoreductase (FNR), hydrogenase (HYDA1), and ferredoxin 5 (FDX5) were quantified in aerobic and anaerobic Chlamydomonas reinhardtii whole cells using purified (recombinant) proteins as internal standards in a mass spectrometric approach. Quantified protein amounts were related to the estimated amount of PSI. The ratios of PC to FNR to HYDA1 to FDX5 in aerobic cells were determined to be 1.4:1.2:0.003:0. In anaerobic cells, the ratios changed to 1.1:1.3:0.019:0.027 (PC:FNR:HYDA1:FDX5). Employing sodium dithionite and methyl viologen as electron donors, the specific activity of hydrogenase in whole cells was calculated to be 382 ± 96.5 μmolH2min-1mg-1. Importantly, these data reveal an about 70-fold lower abundance of HYDA1 compared to FNR. Despite this great disproportion between both proteins, which might further enhance the competition for electrons, the alga is capable of hydrogen production under anaerobic conditions, thus pointing to an efficient channeling mechanism of electrons from FDX1 to the HYDA1.

Flavin-Based Electron Bifurcation, A New Mechanism of Biological Energy Coupling.

There are two types of electron bifurcation (EB), either quinone- or flavin-based (QBEB/FBEB), that involve reduction of a quinone or flavin by a two-electron transfer and two reoxidations by a high- and low-potential one-electron acceptor with a reactive semiquinone intermediate. In QBEB, the reduced low-potential acceptor (cytochrome b) is exclusively used to generate ΔμH+. In FBEB, the "energy-rich" low-potential reduced ferredoxin or flavodoxin has dual function. It can give rise to ΔμH+/Na+ via a ferredoxin:NAD reductase (Rnf) or ferredoxin:proton reductase (Ech) or conducts difficult reductions such as CO2 to CO. The QBEB membrane complexes are similar in structure and function and occur in all domains of life. In contrast, FBEB complexes are soluble and occur only in strictly anaerobic bacteria and archaea (FixABCX being an exception). The FBEB complexes constitute a group consisting of four unrelated families that contain (1) electron-transferring flavoproteins (EtfAB), (2) NAD(P)H dehydrogenase (NuoF homologues), (3) heterodisulfide reductase (HdrABC) or HdrABC homologues, and (4) NADH-dependent ferredoxin:NADP reductase (NfnAB). The crystal structures and electron transport of EtfAB-butyryl-CoA dehydrogenase and NfnAB are compared with those of complex III of the respiratory chain (cytochrome bc1), whereby unexpected common features have become apparent.

Development of a Rubredoxin-Type Center Embedded in a de Dovo-Designed Three-Helix Bundle.

Protein design is a powerful tool for interrogating the basic requirements for the function of a metal site in a way that allows for the selective incorporation of elements that are important for function. Rubredoxins are small electron transfer proteins with a reduction potential centered near 0 mV (vs normal hydrogen electrode). All previous attempts to design a rubredoxin site have focused on incorporating the canonical CXXC motifs in addition to reproducing the peptide fold or using flexible loop regions to define the morphology of the site. We have produced a rubredoxin site in an utterly different fold, a three-helix bundle. The spectra of this construct mimic the ultraviolet-visible, Mössbauer, electron paramagnetic resonance, and magnetic circular dichroism spectra of native rubredoxin. Furthermore, the measured reduction potential suggests that this rubredoxin analogue could function similarly. Thus, we have shown that an α-helical scaffold sustains a rubredoxin site that can cycle with the desired potential between the Fe(II) and Fe(III) states and reproduces the spectroscopic characteristics of this electron transport protein without requiring the classic rubredoxin protein fold.

α-Lactalbumin-oleic acid complex kills tumor cells by inducing excess energy metabolism but inhibiting mRNA expression of the related enzymes

Previous studies have demonstrated that the anti-tumor α-lactalbumin-oleic acid complex (α-LA-OA) may target the glycolysis of tumor cells. However, few data are available regarding the effects of α-LA-OA on energy metabolism. In this study, we measured glycolysis and mitochondrial functions in HeLa cells in response to α-LA-OA using the XF flux analyzer (Seahorse Bioscience, North Billerica, MA). The gene expression of enzymes involved in glycolysis, tricarboxylic acid cycle, electron transfer chain, and ATP synthesis were also evaluated. Our results show that α-LA-OA significantly enhanced the basal glycolysis and glycolytic capacity. Mitochondrial oxidative phosphorylation, including the basal respiration, maximal respiration, spare respiratory capacity and ATP production were also improved in response to α-LA-OA. The enhanced mitochondrial functions maybe partly due to the increased capacity of utilizing fatty acids and glutamine as the substrate. However, the gene expressions of pyruvate kinase M2, lactate dehydrogenase A, aconitate hydratase, and isocitrate dehydrogenase 1 were inhibited, suggesting an insufficient ability for the glycolysis process and the tricarboxylic acid cycle. The increased expression of acetyl-coenzyme A acyltransferase 2, a central enzyme involved in the β-oxidation of fatty acids, would enhance the unbalance due to the decreased expression of electron transfer flavoprotein β subunit, which acts as the electron acceptor. These results indicated that α-LA-OA may induce oxidative stress due to conditions in which the ATP production is exceeding the energy demand. Our results may help clarify the mechanism of apoptosis induced by reactive oxygen species and mitochondrial destruction.

Photoreceptors Take Charge: Emerging Principles for Light Sensing.

The first stage in biological signaling is based on changes in the functional state of a receptor protein triggered by interaction of the receptor with its ligand(s). The light-triggered nature of photoreceptors allows studies on the mechanism of such changes in receptor proteins using a wide range of biophysical methods and with superb time resolution. Here, we critically evaluate current understanding of proton and electron transfer in photosensory proteins and their involvement both in primary photochemistry and subsequent processes that lead to the formation of the signaling state. An insight emerging from multiple families of photoreceptors is that ultrafast primary photochemistry is followed by slower proton transfer steps that contribute to triggering large protein conformational changes during signaling state formation. We discuss themes and principles for light sensing shared by the six photoreceptor families: rhodopsins, phytochromes, photoactive yellow proteins, light-oxygen-voltage proteins, blue-light sensors using flavin, and cryptochromes.

Functional Redundancy in Perchlorate and Nitrate Electron Transport Chains and Rewiring Respiratory Pathways to Alter Terminal Electron Acceptor Preference.

Most dissimilatory perchlorate reducing bacteria (DPRB) are also capable of respiratory nitrate reduction, and preferentially utilize nitrate over perchlorate as a terminal electron acceptor. The similar domain architectures and phylogenetic relatedness of the nitrate and perchlorate respiratory complexes suggests a common evolutionary history and a potential for functionally redundant electron carriers. In this study, we identify key genetic redundancies in the electron transfer pathways from the quinone pool(s) to the terminal nitrate and perchlorate reductases in Azospira suillum PS (hereafter referred to as PS). We show that the putative quinol dehydrogenases, (PcrQ and NapC) and the soluble cytochrome electron carriers (PcrO and NapO) are functionally redundant under anaerobic growth conditions. We demonstrate that, when grown diauxically with both nitrate and perchlorate, the endogenous expression of NapC and NapO during the nitrate reduction phase was sufficient to completely erase any growth defect in the perchlorate reduction phase caused by deletion of pcrQ and/or pcrO. We leveraged our understanding of these genetic redundancies to make PS mutants with altered electron acceptor preferences. Deletion of the periplasmic nitrate reductase catalytic subunit, napA, led to preferential utilization of perchlorate even in the presence of equimolar nitrate, and deletion of the electron carrier proteins napQ and napO, resulted in concurrent reduction of nitrate and perchlorate. Our results demonstrate that nitrate and perchlorate respiratory pathways in PS share key functionally redundant electron transfer proteins and that mutagenesis of these proteins can be utilized as a strategy to alter the preferential usage of nitrate over perchlorate.

Distinct properties underlie flavin-based electron bifurcation in a novel electron transfer flavoprotein FixAB from Rhodopseudomonas palustris

A newly recognized third fundamental mechanism of energy conservation in biology, electron bifurcation, uses free energy from exergonic redox reactions to drive endergonic redox reactions. Flavin-based electron bifurcation furnishes low-potential electrons to demanding chemical reactions, such as reduction of dinitrogen to ammonia. We employed the heterodimeric flavoenzyme FixAB from the diazotrophic bacterium Rhodopseudomonas palustris to elucidate unique properties that underpin flavin-based electron bifurcation. FixAB is distinguished from canonical electron transfer flavoproteins (ETFs) by a second FAD that replaces the AMP of canonical ETF. We exploited near-UV-visible CD spectroscopy to resolve signals from the different flavin sites in FixAB and to interrogate the putative bifurcating FAD. CD aided in assigning the measured reduction midpoint potentials (E° values) to individual flavins, and the E° values tested the accepted model regarding the redox properties required for bifurcation. We found that the higher-E° flavin displays sequential one-electron (1-e-) reductions to anionic semiquinone and then to hydroquinone, consistent with the reactivity seen in canonical ETFs. In contrast, the lower-E° flavin displayed a single two-electron (2-e-) reduction without detectable accumulation of semiquinone, consistent with unstable semiquinone states, as required for bifurcation. This is the first demonstration that a FixAB protein possesses the thermodynamic prerequisites for bifurcating activity, and the separation of distinct optical signatures for the two flavins lays a foundation for mechanistic studies to learn how electron flow can be directed in a protein environment. We propose that a novel optical signal observed at long wavelength may reflect electron delocalization between the two flavins.

Anammox Organism KSU-1 Expresses a Novel His/DOPA Ligated Cytochrome c

Anammox is a bacterial energy metabolic process that forms N2 gas from nitrite and ammonium ions. The enzymatic mechanisms of anammox have been gradually revealed; however, the electron transport chain in anammox bacteria remains poorly understood. In the present study, we purified and characterized two low-molecular-weight c-type cytochromes from an enriched culture of the anammox bacterium strain, KSU-1. Their genes, KSU1_B0428 and KSU1_C0855, were identified in the KSU-1 genome, and their recombinant proteins were characterized. KSU1_B0428 is a typical c-type cytochrome with a His/Met coordinated heme, acting as an electron transfer protein. In contrast, KSU1_C0855 could not be assigned as a known cytochrome and its heme was suggested to have an uncommon axial ligand set. Crystal structural analyses of C0855 clearly showed that its heme iron is coordinated by His15 as a fifth ligand. Moreover, the sixth coordination site is occupied by the aromatic ring of Tyr60, and an unassignable electron density that is inseparable with that of aromatic carbon of Tyr60 was found. The additional electron density was assigned to an O atom by molecular mass analyses. Therefore, Tyr60 would be chemically modified to 3,4-dihydroxyphenylalanine and bound to the Fe atom. We revealed that an anammox bacterium strain KSU-1 expresses a novel cytochrome c having an unprecedented His/3,4-dihydroxyphenylalanine coordinating heme. The expression of the novel c-type cytochrome might be required for the redox reaction of the anammox process.

Irradiation of rainbow trout at early life stages results in a proteomic legacy in adult gills. Part B; the effect of a second radiation dose, after one year, on the proteomic responses in the irradiated and non-irradiated bystander fish

This study extends the investigation of the legacy effects of exposure to a single radiation dose at one of four early life stages, in adult rainbow trout (Part A), by examining the effects of a second identical dose after one year; i.e. egg 48 h after fertilisation (48 h egg) + 1 year, eyed egg + 1 year, yolk sac larvae (YSL) + 1 year and first feeder + 1 year. This included the induction of a bystander effect in non-irradiated trout which had swam with the irradiated fish. The second radiation dose negated any beneficial proteomic responses following early life stage irradiation only, particularly irradiation of 48 h eggs and eyed eggs (Part A). Instead the responses after early life stage + 1 year irradiation are consistently associated with tumorigenesis, cancer progression, or are otherwise damaging: upregulation of alpha-globin 1 (YSL + 1 year and first feeders + 1 year) and downregulation of histone H1, type II keratin, malate dehydrogenase 2–2, Na/K ATPase alpha subunit isoform 1b, nucleoside diphosphate kinase (48 h egg + 1 year), electron transfer flavoprotein subunit alpha (eyed egg + 1 year), 60 S ribosomal protein L30 (YSL + 1 year) and haemoglobin subunit beta-4 (first feeder + 1 year). Most significantly the second radiation dose also negated the overwhelmingly beneficial bystander effect proteomic responses induced by trout irradiated at an early life stage only (Part A). Instead the bystander effect proteomic changes induced by trout irradiated at an early life stage and again at 1 year have been associated with uncertain, with respect to tumorigenesis, or detrimental effects; upregulation of alpha-globin 1 (YSL + 1 year and first feeder + 1 year) and downregulation of malate dehydrogenase 2–2, nucleoside diphosphate kinase (48 h egg + 1 year), transferrin precursor (eyed egg + 1 year), 60 S ribosomal protein L30 (YSL + 1 year) and serine / threonine-protein phosphatase 2 A 65 kDa (first feeder + 1 year). This difference between the bystander effect induced proteomic changes following early life stage irradiation only and early life stage + 1 year irradiation may indicate a fundamental change in the non-targeted effects of radiation following multiple exposure to radiation.

Adjustments to Photosystem Stoichiometry and Electron Transfer Proteins Are Key to the Remarkably Fast Growth of the Cyanobacterium Synechococcus elongatus UTEX 2973.

ABSTRACTAt the genome level, Synechococcus elongatus UTEX 2973 (Synechococcus 2973) is nearly identical to the model cyanobacterium Synechococcus elongatus PCC 7942 (Synechococcus 7942) with only 55 single nucleotide differences separating the two strains. Despite the high similarity between the two strains, Synechococcus 2973 grows three times faster, accumulates significantly more glycogen, is tolerant to extremely high light intensities, and displays higher photosynthetic rates. The high homology between the two strains provides a unique opportunity to examine the factors that lead to increased photosynthetic rates. We compared the photophysiology of the two strains and determined the differences in Synechococcus 2973 that lead to increased photosynthetic rates and the concomitant increase in biomass production. In this study, we identified inefficiencies in the electron transport chain of Synechococcus 7942 that have been alleviated in Synechococcus 2973. Photosystem II (PSII) capacity is the same in both strains. However, Synechococcus 2973 exhibits a 1.6-fold increase in PSI content, a 1.5-fold increase in cytochrome b6f content, and a 2.4-fold increase in plastocyanin content on a per cell basis. The increased content of electron carriers allows a higher flux of electrons through the photosynthetic electron transport chain, while the increased PSI content provides more oxidizing power to maintain upstream carriers ready to accept electrons. These changes serve to increase the photosynthetic efficiency of Synechococcus 2973, the fastest growing cyanobacterium known.

Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria.

Implementing a Statistical Thermodynamic Model to Describe Functionally Relevant Cytochrome C - Cardiolipin L-Site Binding

The L-site binding of ferricytochrome c to cardiolipin (CL) - containing liposomes at slightly acidic pH has attracted interest owing to its potential role in converting this classical electron transfer protein into a peroxidase. Various lines of evidence presented by Nantes and coworkers suggest that this mode of binding is electrostatic, active below pH 7, and involves residues K22, K25, and K27 as well as H26 and H33. We combined several spectroscopic techniques to characterize L-site binding thermodynamically and to identify concomitant structural changes. To this end we employed and extended a recently presented thermodynamic model that describes cytochrome c binding to CL- containing liposomes as a two-step process, where native-like liposome-bound conformers convert to more unfolded conformers with an increase in CL concentration. These partially unfolded species are characterized by a loss of the M80 ligand, which is likely replaced by H26 and/or H33. At acidic pH, the partially unfolded low-spin species converts into a mixture of penta- and hexacoordinate high-spin species at moderate-to- high CL concentration. This change of the heme iron's spin state is most likely caused by the protonation of the non-native histidine ligand, which is facilitated by low effective pH at the liposome surface. Results of our analysis of binding isotherms obtained in the absence and presence of NaCl confirmed the electrostatic character of L-site binding. The inhibiting effect of sodium ions is attributed to the accumulation of cations in the liposome's double layer, which reduces its surface potential. Thus, L-site binding is clearly distinct from cytochrome c - CL interactions at neutral pH where only the conformational change on the membrane surface is affected by the presence of NaCl.

Vacuolar Protein Degradation via Autophagy Provides Substrates to Amino Acid Catabolic Pathways as an Adaptive Response to Sugar Starvation in Arabidopsis thaliana.

The vacuolar lytic degradation of proteins releases free amino acids that plants can use instead of sugars for respiratory energy production. Autophagy is a major cellular process leading to the transport of proteins into the vacuole for degradation. Here, we examine the contribution of autophagy to the amino acid metabolism response to sugar starvation in mature leaves of Arabidopsis thaliana. During sugar starvation arising from the exposure of wild-type (WT) plants to darkness, autophagic transport of chloroplast stroma, which contains most of the proteins in a leaf, into the vacuolar lumen was induced within 2 d. During this time, the level of soluble proteins, primarily Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), decreased and the amount of free amino acid increased. In dark-treated autophagy-defective (atg) mutants, the decrease of soluble proteins was suppressed, which resulted in the compromised release of basic amino acids, branched-chain amino acids (BCAAs) and aromatic amino acids. The impairment of BCAA catabolic pathways in the knockout mutants of the electron transfer flavoprotein (ETF)/ETF:ubiquinone oxidoreductase (etfqo) complex and the electron donor protein isovaleryl-CoA dehydrogenase (ivdh) caused a reduced tolerance to dark treatment similar to that in the atg mutants. The enhanced accumulation of BCAAs in the ivdh and etfqo mutants during the dark treatment was reduced by additional autophagy deficiency. These results indicate that vacuolar protein degradation via autophagy serves as an adaptive response to disrupted photosynthesis by providing substrates to amino acid catabolic pathways, including BCAA catabolism mediated by IVDH and ETFQO.

Role of METTL20 in regulating \u03b2-oxidation and heat production in mice under fasting or ketogenic conditions

METTL20 is a seven-β-strand methyltransferase that is localised to the mitochondria and tri-methylates the electron transfer flavoprotein (ETF) β subunit (ETFB) at lysines 200 and 203. It has been shown that METTL20 decreases the ability of ETF to extract electrons from medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) and glutaryl-CoA dehydrogenase in vitro. METTL20-mediated methylation of ETFB influences the oxygen consumption rate in permeabilised mitochondria, suggesting that METTL20-mediated ETFB methylation may also play a regulatory role in mitochondrial metabolism. In this study, we generated Mettl20 knockout (KO) mice to uncover the in vivo functions of METTL20. The KO mice were viable, and a loss of ETFB methylation was confirmed. In vitro enzymatic assays revealed that mitochondrial ETF activity was higher in the KO mice than in wild-type mice, suggesting that the KO mice had higher β-oxidation capacity. Calorimetric analysis showed that the KO mice fed a ketogenic diet had higher oxygen consumption and heat production. A subsequent cold tolerance test conducted after 24 h of fasting indicated that the KO mice had a better ability to maintain their body temperature in cold environments. Thus, METTL20 regulates ETF activity and heat production through lysine methylation when β-oxidation is highly activated.

Impedance spectroscopy of single bacterial nanofilament reveals water-mediated charge transfer.

For decades respiratory chain and photosystems were the main firing field of the studies devoted to mechanisms of electron transfer in proteins. The concept of conjugated lateral electron and transverse proton transport during cellular respiration and photosynthesis, which was formulated in the beginning of 1960-s, has been confirmed by thousands of experiments. However, charge transfer in recently discovered bacterial nanofilaments produced by various electrogenic bacteria is regarded currently outside of electron and proton conjugation concept. Here we report the new study of charge transfer within nanofilaments produced by Shewanella oneidensis MR-1 conducted in atmosphere of different relative humidity (RH). We utilize impedance spectroscopy and DC (direct current) transport measurements to find out the peculiarities of conductivity and Raman spectroscopy to analyze the nanofilaments’ composition. Data analysis demonstrates that apparent conductivity of nanofilaments has crucial sensitivity to humidity and contains several components including one with unusual behavior which we assign to electron transport. We demonstrate that in the case of Shewanella oneidensis MR-1 charge transfer within these objects is strongly mediated by water. Basing on current data analysis of conductivity we conclude that the studied filaments of Shewanella oneidensis MR-1 are capable of hybrid (conjugated) electron and ion conductivity.

Transferring the entatic-state principle to copper photochemistry.

The entatic state denotes a distorted coordination geometry of a complex from its typical arrangement that generates an improvement to its function. The entatic-state principle has been observed to apply to copper electron-transfer proteins and it results in a lowering of the reorganization energy of the electron-transfer process. It is thus crucial for a multitude of biochemical processes, but its importance to photoactive complexes is unexplored. Here we study a copper complex-with a specifically designed constraining ligand geometry-that exhibits metal-to-ligand charge-transfer state lifetimes that are very short. The guanidine-quinoline ligand used here acts on the bis(chelated) copper(I) centre, allowing only small structural changes after photoexcitation that result in very fast structural dynamics. The data were collected using a multimethod approach that featured time-resolved ultraviolet-visible, infrared and X-ray absorption and optical emission spectroscopy. Through supporting density functional calculations, we deliver a detailed picture of the structural dynamics in the picosecond-to-nanosecond time range.