Electron Transfer Proteins Research Articles

Positional Distributions of the Tethered Modules in Nitric Oxide Synthase: Monte Carlo Calculations and Pulsed EPR Measurements.

The nitric oxide synthase (NOS) enzyme consists of multiple domains connected by flexible random coil tethers. In a catalytic cycle, the NOS domains move within the limits determined by the length and flexibility of the interdomain tethers and form docking complexes with each other. This process represents a key component of the electron transport from the flavin adenine dinucleotide/reduced nicotinamide adenine dinucleotide phosphate binding domain to the catalytic heme centers located in the oxygenase domain. Studying the conformational behavior of NOS is therefore imperative for a full understanding of the overall catalytic mechanism. In this work, we have investigated the equilibrium positional distributions of the NOS domains and the bound calmodulin (CaM) by using Monte Carlo calculations of the NOS conformations. As a main experimental reference, we have used the magnetic dipole interaction between a bifunctional spin label attached to T34C/S38C mutant CaM and the NOS heme centers, which was measured by pulsed electron paramagnetic resonance. In general, the calculations of the conformational distributions allow one to determine the range and statistics of positions occupied by the tethered protein domains, assess the crowding effect of the multiple domains on each other, evaluate the accessibility of various potential domain docking sites, and estimate the interaction energies required to achieve target populations of the docked states. In the particular application described here, we have established the specific mechanisms by which the bound CaM facilitates the flavin mononucleotide (FMN)/heme interdomain docking in NOS. We have also shown that the intersubunit FMN/heme domain docking and electron transfer in the homodimeric NOS protein are dictated by the existing structural makeup of the protein. Finally, from comparison of the calculated and experimental docking probabilities, the characteristic stabilization energies for the CaM/heme domain and the FMN domain/heme domain docking complexes have been estimated as -4.5kT and -10.5kT, respectively.

Raman Evidence of p53-DBD Disorder Decrease upon Interaction with the Anticancer Protein Azurin.

Raman spectroscopy, which is a suitable tool to elucidate the structural properties of intrinsically disordered proteins, was applied to investigate the changes in both the structure and the conformational heterogeneity of the DNA-binding domain (DBD) belonging to the intrinsically disordered protein p53 upon its binding to Azurin, an electron-transfer anticancer protein from Pseudomonas aeruginosa. The Raman spectra of the DBD and Azurin, isolated in solution or forming a complex, were analyzed by a combined analysis based on peak inspection, band convolution, and principal component analysis (PCA). In particular, our attention was focused on the Raman peaks of Tyrosine and Tryptophan residues, which are diagnostic markers of protein side chain environment, and on the Amide I band, of which the deconvolution allows us to extract information about α-helix, β-sheet, and random coil contents. The results show an increase of the secondary structure content of DBD concomitantly with a decrease of its conformational heterogeneity upon its binding to Azurin. These findings suggest an Azurin-induced conformational change of DBD structure with possible implications for p53 functionality.

Effect of Macromolecular Crowding on the FMN-Heme Intraprotein Electron Transfer in Inducible NO Synthase.

Previous biochemical studies of nitric oxide synthase enzymes (NOSs) were conducted in diluted solutions. However, the intracellular milieu where the proteins perform their biological functions is crowded with macromolecules. The effect of crowding on the electron transfer kinetics of multidomain proteins is much less understood. Herein, we investigated the effect of macromolecular crowding on the FMN-heme intraprotein interdomain electron transfer (IET), an obligatory step in NOS catalysis. A noticeable increase in the IET rate in the bidomain oxygenase/FMN (oxyFMN) and the holoprotein of human inducible NOS (iNOS) was observed upon addition of Ficoll 70 in a nonsaturable manner. Additionally, the magnitude of IET enhancement for the holoenzyme is much higher than that that of the oxyFMN construct. The crowding effect is also evident at different ionic strengths. Importantly, the enhancing extent is similar for the iNOS oxyFMN protein with added Ficoll 70 and Dextran 70 that give the same solution viscosity, showing that specific interactions do not exist between the NOS protein and the crowder. Moreover, the population of the docked FMN-heme state is significantly increased upon addition of Ficoll 70 and the fluorescence lifetime values do not correspond to those in the absence of Ficoll 70. The steady-state cytochrome c reduction by the holoenzyme is noticeably enhanced by the crowder, while the ferricyanide reduction is unchanged. The NO production activity of the iNOS holoenzyme is stimulated by Ficoll 70. The effect of macromolecular crowding on the kinetics can be rationalized on the basis of the excluded volume effect, with an entropic origin. The intraprotein electron transfer kinetics, fluorescence lifetime, and steady-state enzymatic activity results indicate that macromolecular crowding modulates the NOS electron transfer through multiple pathways. Such a mechanism should be applicable to electron transfer in other multidomain redox proteins.

An acyl-CoA dehydrogenase microplate activity assay using recombinant porcine electron transfer flavoprotein

Acyl-CoA dehydrogenases (ACADs) play key roles in the mitochondrial catabolism of fatty acids and branched-chain amino acids. All nine characterized ACAD enzymes use electron transfer flavoprotein (ETF) as their redox partner. The gold standard for measuring ACAD activity is the anaerobic ETF fluorescence reduction assay, which follows the decrease of pig ETF fluorescence as it accepts electrons from an ACAD in vitro. Although first described 35 years ago, the assay has not been widely used due to the need to maintain an anaerobic assay environment and to purify ETF from pig liver mitochondria. Here, we present a method for expressing recombinant pig ETF in E coli and purifying it to homogeneity. The recombinant protein is virtually pure after one chromatography step, bears higher intrinsic fluorescence than the native enzyme, and provides enhanced activity in the ETF fluorescence reduction assay. Finally, we present a simplified protocol for removing molecular oxygen that allows adaption of the assay to a 96-well plate format. The availability of recombinant pig ETF and the microplate version of the ACAD activity assay will allow wide application of the assay for both basic research and clinical diagnostics.

Thermodynamic View of Proton Activated Electron Transfer in the Reaction Center of Photosynthetic Bacteria.

The temperature dependence of the sequential coupling of proton transfer to the second interquinone electron transfer is studied in the reaction center proteins of photosynthetic bacteria modified by different mutations and treatment by divalent cations. The Eyring plots of kinetics were evaluated by the Marcus theory of electron and proton transfer. In mutants of electron transfer limitation (including the wild type), the observed thermodynamic parameters had to be corrected for those of the fast proton pre-equilibrium. The electron transfer is nonadiabatic with transmission coefficient 6 × 10-4, and the reorganization energy amounts to 1.2 eV. If the proton transfer is the rate limiting step, the reorganization energy and the works terms fall in the range of 200-500 meV, depending on the site of damage in the proton transfer chain. The product term is 100-150 meV larger than the reactant term. While the electron transfer mutants have a low free energy of activation (∼200 meV), the proton transfer variants show significantly elevated levels of the free energy barrier (∼500 meV). The second electron transfer in the bacterial reaction center can serve as a model system of coupled electron and proton transfer in other proteins or ion channels.

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 β‐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 k catapp/K Mapp 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.

6-Hydroxypseudooxynicotine Dehydrogenase Delivers Electrons to Electron Transfer Flavoprotein during Nicotine Degradation by Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways. The hybrid pathway consists of at least six steps involved in oxidoreductive reactions before the N-heterocycle can be broken down. Collectively, the six steps allow electron transfer from nicotine and its intermediates to the final acceptor O2 via the electron transport chain (ETC). 6-Hydroxypseudooxynicotine oxidase, renamed 6-hydroxypseudooxynicotine dehydrogenase in this study, has been characterized as catalyzing the fourth step using the artificial electron acceptor 2,6-dichlorophenolindophenol. Here, we used biochemical, genetic, and liquid chromatography-mass spectrometry (LC-MS) analyses to determine that 6-hydroxypseudooxynicotine dehydrogenase utilizes the electron transfer flavoprotein (EtfAB) as the physiological electron acceptor to catalyze the dehydrogenation of pseudooxynicotine, an analogue of the true substrate 6-hydroxypseudooxynicotine, in vivo, into 3-succinoyl-semialdehyde-pyridine. NAD(P)+, O2, and ferredoxin could not function as electron acceptors. The oxygen atom in the aldehyde group of the product 3-succinoyl-semialdehyde-pyridine was verified to be derived from H2O. Disruption of the etfAB genes in the nicotine-degrading gene cluster decreased the growth rate of A. tumefaciens S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine, an intermediate downstream of the hybrid pathway, indicating the requirement of EtfAB for efficient nicotine degradation. The electrons were found to be further transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase. These results aid in an in-depth understanding of the electron transfer process and energy metabolism involved in the nicotine oxidation and provide novel insights into nicotine catabolism in bacteria.IMPORTANCE Nicotine has been studied as a model for toxic N-heterocyclic aromatic compounds. Microorganisms can catabolize nicotine via various pathways and conserve energy from its oxidation. Although several oxidoreductases have been characterized to participate in nicotine degradation, the electron transfer involved in these processes is poorly understood. In this study, we found that 6-hydroxypseudooxynicotine dehydrogenase, a key enzyme in the hybrid pyridine and pyrrolidine pathway for nicotine degradation in Agrobacterium tumefaciens S33, utilizes EtfAB as a physiological electron acceptor. Catalyzed by the membrane-associated electron transfer flavoprotein:ubiquinone oxidoreductase, the electrons are transferred from the reduced EtfAB to coenzyme Q, which then could enter into the classic ETC. Thus, the route for electron transport from the substrate to O2 could be constructed, by which ATP can be further sythesized via chemiosmosis to support the baterial growth. These findings provide new knowledge regarding the catabolism of N-heterocyclic aromatic compounds in microorganisms.

Structural Factors for Regulation of Electron Transfer from Cytochrome C to Cytochrome C Oxidase

The electron transfer (ET) reaction from cytochrome c (Cyt c) to cytochrome c oxidase (CcO) is the terminal ET reaction in the respiratory chain of mitochondria. By accepting four electrons from the repetitive binding of Cyt c, CcO reduces molecular oxygen to water molecules to promote the proton pumping from the matrix to inner membrane space in mitochondria, resulting in the formation of the proton gradient, which is the primary driving force for the ATP synthesis in the complex V. The ET reaction from Cyt c to CcO is, therefore, the crucial step for the energy generation system in the cellular level. Because the incomplete reduction of molecular oxygen generates reactive oxygen species, which is highly cytotoxic to damage cellular components, the ET reaction from Cyt c to CcO is thought to be strictly regulated and the formation of the specific ET pathway from Cyt c to CcO in the ET complex has been suggested. However, the ET pathway and its regulation mechanism are still controversial due to the lack of the structural information about the ET complex between Cyt c and CcO. To determine the structure of the Cyt c – CcO ET complex, we used the protein-protein docking simulation1 between Cyt c and CcO based on the NMR structural data for the interaction site for CcO on Cyt c 2. The predicted complex structure between Cyt c and CcO clearly showed that the interaction site between the two proteins is highly hydrophobic and the mutational study also revealed that the introduction of hydrophilic amino acid residues significantly reduced the ET rate from Cyt c to CcO3. The formation of such a hydrophobic interaction site was confirmed by the osmotic pressure experiments for the steady-state kinetic measurements of the ET reaction from Cyt c to CcO. Based on the osmotic pressure dependence of Michaelis constant, K m, for the ET reaction from Cyt c to CcO, we found out the significant dehydration from hydrophobic amino acid residues near the exposed hydrophobic heme periphery, facing to the hydrophobic amino acid residues of CcO, to form the hydrophobic interface between Cyt c and CcO in the predicted ET complex. The protein-protein docking simulation for the complex formation with a mutant having the hydrophilic amino acid residues in the interaction site for CcO indicated that a new hydrogen bond was formed in the interaction site between Cyt c and CcO, implying that the incomplete dehydration in the interaction site and/or invasion of the bulk water to the interaction site of the mutant. To determine the ET pathway from Cyt c to CcO, the conventional ET pathway analysis4 was applied to the predicted ET complex between Cyt c and CcO. Although the estimated ET pathway was constructed by many hydrophobic amino acid residues, the introduction of the hydrophobic amino acid residue into the interaction site resulted in the accelerated ET rate in the pathway analysis, which is inconsistent with the experimental data. We re-evaluate the ET pathway using the Green’s function and found that electron is directly transferred from the heme iron of Cyt c to the cupper ion of the subunit II of CcO through the tunneling mechanism, and the decreased hydrophobicity in the interaction site reduced the ET rate in this analysis. We, therefore, conclude that the formation of the hydrophobic interaction site by the dehydration around exposed heme periphery is essential for the regulation of the ET reaction from Cyt c to CcO in the respiratory chain. Sato, W.; Hitaoka, S.; Inoue, K.; Imai, M.; Saio, T.; Uchida, T.; Shinzawa-Itoh, K.; Yoshikawa, S.; Yoshizawa, K.; Ishimori, K., Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation. J. Biol. Chem. 2016, 291 (29), 15320-15331. Sakamoto, K.; Kamiya, M.; Imai, M.; Shinzawa-Itoh, K.; Uchida, T.; Kawano, K.; Yoshikawa, S.; Ishimori, K., NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase. Proc. Natl. Acad. Sci. USA 2011, 108 (30), 12271-12276. Sato, W.; Uchida, T.; Saio, T.; Ishimori, K., Polyethylene glycol promotes autoxidation of cytochrome c. Biochim. Biophys. Acta 2018, 1862 (6), 1339-1349. Onuchic, J. N.; Beratan, D. N.; Winkler, J. R.; Gray, H. B., Pathway Analysis of Protein Electron-Transfer Reactions. Ann. Rev. Biophys. Biomol. Struct. 1992, 21 (1), 349-377.

Reactivity of [Fe4S4] Clusters toward C1 Substrates: Mechanism, Implications, and Potential Applications.

FeS proteins are metalloproteins prevalent in the metabolic pathways of most organisms, playing key roles in a wide range of essential cellular processes. A member of this protein family, the Fe protein of nitrogenase, is a homodimer that contains a redox-active [Fe4S4] cluster at the subunit interface and an ATP-binding site within each subunit. During catalysis, the Fe protein serves as the obligate electron donor for its catalytic partner, transferring electrons concomitant with ATP hydrolysis to the cofactor site of the catalytic component to enable substrate reduction. The effectiveness of Fe protein in electron transfer is reflected by the unique reactivity of nitrogenase toward small-molecule substrates. Most notably, nitrogenase is capable of catalyzing the ambient reduction of N2 and CO into NH4+ and hydrocarbons, respectively, in reactions that parallel the important industrial Haber-Bosch and Fischer-Tropsch processes. Other than participating in nitrogenase catalysis, the Fe protein also functions as an essential factor in nitrogenase assembly, which again highlights its capacity as an effective, ATP-dependent electron donor. Recently, the Fe protein of a soil bacterium, Azotobacter vinelandii, was shown to act as a reductase on its own and catalyze the ambient conversion of CO2 to CO at its [Fe4S4] cluster either under in vitro conditions when a strong reductant is supplied or under in vivo conditions through the action of an unknown electron donor(s) in the cell. Subsequently, the Fe protein of a mesophilic methanogenic organism, Methanosarcina acetivorans, was shown to catalyze the in vitro reduction of CO2 and CO into hydrocarbons under ambient conditions, illustrating an impact of protein scaffold on the redox properties of the [Fe4S4] cluster and the reactivity of the cluster toward C1 substrates. This reactivity was further traced to the [Fe4S4] cluster itself, as a synthetic [Fe4S4] compound was shown to catalyze the reduction of CO2 and CO to hydrocarbons in solutions in the presence of a strong reductant. Together, these observations pointed to an inherent ability of the [Fe4S4] clusters and, possibly, the FeS clusters in general to catalyze C1-substrate reduction. Theoretical calculations have led to the proposal of a plausible reaction pathway that involves the formation of hydrocarbons via aldehyde-like intermediates, providing an important framework for further mechanistic investigations of FeS-based activation and reduction of C1 substrates. In this Account, we summarize the recent work leading to the discovery of C1-substrate reduction by protein-bound and free [Fe4S4] clusters as well as the current mechanistic understanding of this FeS-based reactivity. In addition, we briefly discuss the evolutionary implications of this discovery and potential applications that could be developed to enable FeS-based strategies for the ambient recycling of unwanted C1 waste into useful chemical commodities.

Structural Changes in Adsorbed Cytochrome c upon Applied Potential Characterized by Neutron Reflectometry.

The structural behavior of an electron-transfer protein, cytochrome c, at the 316L stainless steel electrode/aqueous interface was investigated over a range of applied potentials using neutron reflectometry supported by solution depletion isotherms, X-ray reflectometry, and quartz crystal microbalance measurements. A custom-made electrochemical cell allowed in situ observation of the adsorbed protein across a range of applied potentials; models fitted to the NR data showed a compact inner protein layer at the metal/electrolyte interface and a further thicker but highly diffuse layer that could be removed by rinsing. The overall amount adsorbed was found to be strongly dependent on the applied potential and buffer pH. Subtle but significant changes in the structure of the adsorbed protein layer were seen as the potential was swept between ±0.40 V, reflecting changing attractive/repulsive interactions between the protein's charged side groups and the surface. At greater applied potentials, irreversible changes in the stainless steel film structure were also observed and attributed to deuterium absorption into the metal.

MoIVD-Mediated Leucine Catabolism Is Required for Vegetative Growth, Conidiation and Full Virulence of the Rice Blast Fungus Magnaporthe oryzae.

Isovaleryl-CoA dehydrogenase (IVD), a member of the acyl-CoA dehydrogenase (ACAD) family, is a key enzyme catalyzing the conversion of isovaleryl-CoA to β-methylcrotonyl-CoA in the third reaction of the leucine catabolism pathway and simultaneously transfers electrons to the electron-transferring flavoprotein (ETF) for ATP synthesis. We previously identified the ETF ortholog in rice blast fungus Magnaporthe oryzae (MoETF) and showed that MoETF was essential for fungal growth, conidiation and pathogenicity. To further investigate the biological function of electron-transferring proteins and clarify the role of leucine catabolism in growth and pathogenesis, we characterized MoIVD (M. oryzae isovaleryl-CoA dehydrogenase). MoIvd is highly conserved in fungi and its expression was highly induced by leucine. The Δmoivd mutants showed reduced growth, decreased conidiation and compromised pathogenicity, while the conidial germination and appressorial formation appeared normal. Consistent with a block in leucine degradation, the Δmoivd mutants accumulated isovaleric acid, grew more slowly, fully lacked pigmentation and completely failed to produce conidia on leucine-rich medium. These defects were largely rescued by raising the extracellular pH, suggesting that the accumulation of isovaleric acid contributes to the growth and conidiation defects. However, the reduced virulence of the mutants was probably due to their inability to overcome oxidative stress, since a large amount of ROS (reactive oxygen species) accumulated in infected host cell. In addition, MoIvd is localized to mitochondria and interacted with its electron receptor MoEtfb, the β subunit of MoEtf. Taken together, our results suggest that MoIVD functions in leucine catabolism and is required for the vegetative growth, conidiation and full virulence of M. oryzae, providing the first evidence for IVD-mediated leucine catabolism in the development and pathogenesis of plant fungal pathogens.

The catalytic mechanism of electron-bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD+

Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR, and optical titrations with NADH or inorganic reductants with and without NAD+, we propose a catalytic cycle involving formation of an intermediary NAD+-bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low- and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically unrelated bifurcating NADH-dependent ferredoxin NADP+ oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD+, the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and the two two-electron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETFs and can be applied to the large bifurcating ETF family.

Electrophotonics: multimodal sensors for bacteria identification and phenotyping

A comprehensive understanding of bacteria phenotypes requires tools that are able to characterise structure and function across multiple length scales, from communities and individual cells down to single molecules. Multimodal sensing combines multiple transduction technologies in parallel to probe different properties simultaneously, and thereby increase the range of measurable interactions, the amount of information that can be extracted, and improve detection accuracy. Electrophotonics is a multimodal sensing approach that combines electrochemical and photonic techniques in a single, integrated device that provides enhanced quantitative measurements of chemical reactions. We present a new electrophotonic device based on a Si3N4 guided mode resonant (GMR) structure with an integrated indium tin oxide (ITO) electrode. The GMR structure is sensitive to refractive index changes at the sensor surface, enabling label free, real time detection of biomolecules, microorganisms and imaging of molecular interactions with micrometre-scale resolution to provide spatial information about surface binding interactions. The ITO electrode has been shown to be compatible with voltammetry-based techniques for interrogating redox behaviour along with electrochemical impedance spectroscopy. We demonstrate the wide range of microbiological applications of electrophotonic technology including the characterisation of redox active protein electron transfer and surface adsorption, bacterial adherence and growth on chemically functionalised surfaces, and label free parallel detection of clinically relevant biomarkers. We believe that the multimodal measurements with this novel technology can provide new approaches to investigate and understand microbial biology.

Chronic Hypoxia Enhances β-Oxidation-Dependent Electron Transport via Electron Transferring Flavoproteins.

Hypoxia poses a stress to cells and decreases mitochondrial respiration, in part by electron transport chain (ETC) complex reorganization. While metabolism under acute hypoxia is well characterized, alterations under chronic hypoxia largely remain unexplored. We followed oxygen consumption rates in THP-1 monocytes during acute (16 h) and chronic (72 h) hypoxia, compared to normoxia, to analyze the electron flows associated with glycolysis, glutamine, and fatty acid oxidation. Oxygen consumption under acute hypoxia predominantly demanded pyruvate, while under chronic hypoxia, fatty acid- and glutamine-oxidation dominated. Chronic hypoxia also elevated electron-transferring flavoproteins (ETF), and the knockdown of ETF–ubiquinone oxidoreductase lowered mitochondrial respiration under chronic hypoxia. Metabolomics revealed an increase in citrate under chronic hypoxia, which implied glutamine processing to α-ketoglutarate and citrate. Expression regulation of enzymes involved in this metabolic shunting corroborated this assumption. Moreover, the expression of acetyl-CoA carboxylase 1 increased, thus pointing to fatty acid synthesis under chronic hypoxia. Cells lacking complex I, which experienced a markedly impaired respiration under normoxia, also shifted their metabolism to fatty acid-dependent synthesis and usage. Taken together, we provide evidence that chronic hypoxia fuels the ETC via ETFs, increasing fatty acid production and consumption via the glutamine-citrate-fatty acid axis.

Donor-acceptor symmetric and antisymmetric tunneling matrix elements: a pathway model investigation of protein electron transfer.

In protein electron transfer reaction rate calculations, the electronic Hamiltonian is apportioned into donor-acceptor (D-A) and protein bridge subspaces, and a two-state system is defined for the D-A subspace. Löwdin partitioning is used to perform the two-state reductions necessary to compute the tunneling matrix element between D and A sites. Here, a method of performing donor and acceptor state analysis for a non-orthogonal basis set in both the weak and strong electronic coupling regimes is developed. The electron tunneling current and coupling are obtained in terms of D-A symmetric and antisymmetric interatomic tunneling elements, and are then used to compare pathway models. These interatomic tunneling elements are both proportional to the Green's function elements of the isolated protein bridge. To facilitate a perturbative treatment of antisymmetric interatomic tunneling currents, we found a well-known expression for the D-A tunneling matrix element in terms of transformed Green's function matrix elements of the isolated protein bridge. Also, the relationship of the tunneling matrix element to BO pathways is discussed using the symmetric interatomic coupling. Finally, the definition of the average interatomic and atomic pathway coupling allows us obtain the quantum interference between interatomic tunneling pathways.

Cell factories converting lactate and acetate to butyrate: Clostridium butyricum and microbial communities from dark fermentation bioreactors

BackgroundInteractions between microorganisms during specific steps of anaerobic digestion determine metabolic pathways in bioreactors and consequently the efficiency of fermentation processes. This study focuses on conversion of lactate and acetate to butyrate by bacteria of dark fermentation. The recently recognized flavin-based electron bifurcation as a mode of energy coupling by anaerobes increases our knowledge of anaerobic lactate oxidation and butyrate formation.ResultsMicrobial communities from dark fermentation bioreactors or pure culture of Clostridium butyricum are able to convert lactate and acetate to butyrate in batch experiments. The ability of C. butyricum to transform lactate and acetate to butyrate was shown for the first time, with ethanol identified as an additional end product of this process. A search for genes encoding EtfAB complexes and their gene neighbourhood in C. butyricum and other bacteria capable of lactate and acetate conversion to butyrate as well as butyrate-producers only and the lactate oxidiser Acetobacterium woodii, revealed that the Etf complexes involved in (i) lactate oxidation and (ii) butyrate synthesis, form separate clusters. There is a more extent similarity between Etf subunits that are involved in lactate oxidation in various species (e.g. A. woodii and C. butyricum) than between the different etf gene products within the same species of butyrate producers. A scheme for the metabolic pathway of lactate and acetate transformation to butyrate in C. butyricum was constructed.ConclusionsStudies on the conversion of lactate and acetate to butyrate by microbial communities from dark fermentation bioreactors or Clostridium butyricum suggest that a phenomenon analogous to cross-feeding of lactate in gastrointestinal tract also occurs in hydrogen-yielding reactors. A scheme of lactate and acetate transformation pathway is proposed, based on the example of C. butyricum, which employs flavin-based electron bifurcation. This process utilizes electron-transferring flavoprotein (Etf) complexes specific for (i) lactate oxidation and (ii) butyrate formation. Phylogenetic analysis revealed that such complexes are encoded in the genomes of other bacteria capable of lactate and acetate conversion to butyrate. These findings contribute significantly to our understanding of the metabolic pathways and symbiotic interactions between bacteria during the acidogenic step of anaerobic digestion.

Heterogeneous and Highly Dynamic Interface in Plastocyanin-Cytochrome f Complex Revealed by Site-Specific 2D-IR Spectroscopy.

Transient protein complexes are crucial for sustaining dynamic cellular processes. The complexes of electron-transfer proteins are a notable example, such as those formed by plastocyanin (Pc) and cytochrome f (cyt f) in the photosynthetic apparatus. The dynamic and heterogeneous nature of these complexes, however, makes their study challenging. To better elucidate the complex of Nostoc Pc and cyt f, 2D-IR spectroscopy coupled to site-specific labeling with cyanophenylalanine infrared (IR) probes was employed to characterize how the local environments at sites along the surface of Pc were impacted by cyt f binding. The results indicate that Pc most substantially engages with cyt f via the hydrophobic patch around the copper redox site. Complexation with cyt f led to an increase in inhomogeneous broadening of the probe absorptions, reflective of increased heterogeneity of interactions with their environment. Notably, most of the underlying states interconverted very rapidly (1 to 2 ps), suggesting a complex with a highly mobile interface. The data support a model of the complex consisting of a large population of an encounter complex. Additionally, the study demonstrates the application of 2D-IR spectroscopy with site-specifically introduced probes to reveal new quantitative insight about dynamic biochemical systems.

Why Are DNA and Protein Electron Transfer So Different?

The corpus of electron transfer (ET) theory provides considerable power to describe the kinetics and dynamics of electron flow at the nanoscale. How is it, then, that nucleic acid (NA) ET continues to surprise, while protein-mediated ET is relatively free of mechanistic bombshells? I suggest that this difference originates in the distinct electronic energy landscapes for the two classes of reactions. In proteins, the donor/acceptor-to-bridge energy gap is typically several-fold larger than in NAs. NA ET can access tunneling, hopping, and resonant transport among the bases, and fluctuations can enable switching among mechanisms; protein ET is restricted to tunneling among redox active cofactors and, under strongly oxidizing conditions, a few privileged amino acid side chains. This review aims to provide conceptual unity to DNA and protein ET reaction mechanisms. The establishment of a unified mechanistic framework enabled the successful design of NA experiments that switch electronic coherence effects on and off for ET processes on a length scale of multiple nanometers and promises to provide inroads to directing and detecting charge flow in soft-wet matter.

Clinical and muscle magnetic resonance image findings in patients with late-onset multiple acyl-CoA dehydrogenase deficiency.

Background:Late-onset multiple acyl-coA dehydrogenase deficiency (MADD) is an autosomal recessive inherited metabolic disorder. It is still unclear about the muscle magnetic resonance image (MRI) pattern of the distal lower limb pre- and post-treatment in patients with late-onset MADD. This study described the clinical and genetic findings in a cohort of patients with late-onset MADD, and aimed to characterize the MRI pattern of the lower limbs.Methods:Clinical data were retrospectively collected from clinic centers of Peking University People's Hospital between February 2014 and February 2018. Muscle biopsy, blood acylcarnitines, and urine organic acids profiles, and genetic analysis were conducted to establish the diagnosis of MADD in 25 patients. Muscle MRI of the thigh and leg were performed in all patients before treatment. Eight patients received MRI re-examinations after treatment.Results:All patients presented with muscle weakness or exercise intolerance associated with variants in the electron transfer flavoprotein dehydrogenase gene. Muscle MRI showed a sign of both edema-like change and fat infiltration selectively involving in the soleus (SO) but sparing of the gastrocnemius (GA) in the leg. Similar sign of selective involvement of the biceps femoris longus (BFL) but sparing of the semitendinosus (ST) was observed in the thigh. The sensitivity and specificity of the combination of either “SO+/GA–” sign or “BFL+/ST–” sign for the diagnosis of late-onset MADD were 80.0% and 83.5%, respectively. Logistic regression model supported the findings. The edema-like change in the SO and BFL muscles were quickly recovered at 1 month after treatment, and the clinical symptom was also relieved.Conclusions:This study expands the clinical and genetic spectrums of late-onset MADD. Muscle MRI shows a distinct pattern in the lower limb of patients with late-onset MADD. The dynamic change of edema-like change in the affected muscles might be a potential biomarker of treatment response.

FAD-sequestering proteins protect mycobacteria against hypoxic and oxidative stress

The ability to persist in the absence of growth triggered by low oxygen levels is a critical process for the survival of mycobacterial species in many environmental niches. MSMEG_5243 (fsq), a gene of unknown function in Mycobacterium smegmatis, is up-regulated in response to hypoxia and regulated by DosRDosS/DosT, an oxygen- and redox-sensing two-component system that is highly conserved in mycobacteria. In this communication, we demonstrate that MSMEG_5243 is a flavin-sequestering protein and henceforth refer to it as Fsq. Using an array of biochemical and structural analyses, we show that Fsq is a member of the diverse superfamily of flavin- and deazaflavin-dependent oxidoreductases (FDORs) and is widely distributed in mycobacterial species. We created a markerless deletion mutant of fsq and demonstrate that fsq is required for cell survival during hypoxia. Using fsq deletion and overexpression, we found that fsq enhances cellular resistance to hydrogen peroxide treatment. The X-ray crystal structure of Fsq, solved to 2.7 Å, revealed a homodimeric organization with FAD bound noncovalently. The Fsq structure also uncovered no potential substrate-binding cavities, as the FAD is fully enclosed, and electrochemical studies indicated that the Fsq:FAD complex is relatively inert and does not share common properties with electron-transfer proteins. Taken together, our results suggest that Fsq reduces the formation of reactive oxygen species (ROS) by sequestering free FAD during recovery from hypoxia, thereby protecting the cofactor from undergoing autoxidation to produce ROS. This finding represents a new paradigm in mycobacterial adaptation to hypoxia.