Alternative Electron Transfer Research Articles

Electric wiring of bacteria using redox polymers and selective measurement of metabolic activity in the presence of surrounding planktonic bacteria

Non-electroactive bacteria (n-EAB), constituting the majority of known bacteria to date, have been underutilized in electrochemical conversion technologies due to their lack of direct electron transfer to electrodes. In this study, we established an electric wiring between n-EAB (gram-positive Bacillus subtilis and gram-negative Escherichia coli) and an extracellular electrode via a ferrocene-polyethyleneimine-based redox polymer (Fc-PEI). Chronoamperometry recordings indicated that Fc-PEI can transfer intracellular electrons to the extracellular electrode regardless of the molecular organization of PEI (linear or branched) and the membrane structure of bacteria (gram-positive or -negative). As fluorescence staining suggested, Fc-PEI improves the permeability of the bacterial cell membrane, enabling electron carriers in the cell to react with Fc. In addition, experiments with Fc-immobilized electrodes without PEI suggested the existence of an alternative electron transfer pathway from B. subtilis to the extracellular Fc adsorbed onto the cell membrane. Furthermore, we proposed for the first time that the bacteria/Fc-linear PEI modified structure enables selective measurement of immobilized bacterial activity by physically blocking contact between the electrode surface and planktonic cells co-existing in the surrounding media. Such electrodes can be a powerful analytical tool for elucidating the metabolic activities of specific bacteria wired to the electrode even within complex bacterial communities.

Room temperature preparation of novel g-C3N4/RuNP/ZIF-8 Z-scheme heterojunction for solar-light driven H2 generation

We present a novel approach for the room temperature synthesis and integration of Ru nanoparticles into a Z-scheme heterojunction comprising graphitic carbon nitride (CN) and zeolitic imidazolate framework-8 (ZIF-8). This heterojunction demonstrates outstanding efficiency in sunlight-driven hydrogen (H2) generation via water splitting. Various structural and spectroscopic analyses elucidate Ru nanoparticles' successful incorporation and synergistic effects within the CN/ZIF-8 heterojunction because the decrease in interplanar spacing due to π-π stacking interactions between aromatic rings of tri-s-triazine in CN and imidazole rings in ZIF-8. The Z-scheme configuration facilitates efficient charge separation and promotes H2 production. Additionally, Ru nanoparticles offer alternative electron transfer pathways, enhancing carrier separation and prolonging the lifespan of photoinduced charges compared to CN and ZIF-8 alone. Notably, RZCN-2 heterojunction exhibited significantly enhanced hydrogen production, reaching 5245 μmol g−1 h−1. By analyzing work functions and energy band structures, we demonstrate that the strong interfacial electric fields across the Z-scheme heterojunction effectively mitigate the recombination of photogenerated charges while maintaining the necessary redox capacity for efficient H2 production. The room temperature synthesis and integration of Ru nanoparticles provide a practical and effective strategy for optimizing photocatalytic activity and promoting sustainable energy conversion, thereby advancing photocatalytic technologies.

Long-Range Electron Transport Rates Depend on Wire Dimensions in Cytochrome Nanowires.

The ability to redirect electron transport to new reactions in living systems opens possibilities to store energy, generate new products, or probe physiological processes. Recent work by Huang et al. showed that 3D crystals of small tetraheme cytochromes (STC) can transport electrons over nanoscopic to mesoscopic distances by an electron hopping mechanism, making them promising materials for nanowires. However, fluctuations at room temperature may distort the nanostructure, hindering efficient electron transport. Classical molecular dynamics simulations of these fluctuations at the nano- and mesoscopic scales allowed us to develop a graph network representation to estimate maximum electron flow that can be driven through STC wires. In longer nanowires, transient structural fluctuations at protein-protein interfaces tended to obstruct efficient electron transfer, but these blockages are ameliorated in thicker crystals where alternative electron transfer pathways become more efficient. The model implies that more flexible proteinprotein interfaces limit the required minimum diameter to carry currents commensurate with conventional electronics.

Chlororespiration as a Protective Stress-inducible Electron Transport Pathway in Chloroplasts

Chlororespiration is the uptake of oxygen into the respiratory electron transport chain (ETC) localized in the thylakoid membranes of chloroplasts. The chlororespiratory ETC interacts with photosynthetic electron transport and participates in the non-photochemical reduction/oxidation of the plastoquinone pool (PQP) accompanied by O2 consumption. The two key thylakoid enzymes in chlororespiration are the plastid-encoded NAD(P)H dehydrogenase complex (NDH) and the nucleus-encoded terminal plastoquinol oxidase (PTOX). The contribution of chlororespiratory electron flux to the total electron flow in non-stressed plants is considered insignificant. In contrast, under abiotic stresses, chlororespiration appears to be triggered, at least in some photosynthetic organisms, acting as a protective alternative electron transport pathway. There is evidence of NDH complex and PTOX increasing their activity and/or abundance when plants experience high light, drought, heat, or low-temperature stresses. Alternative electron transfer to oxygen via PTOX protects PQP from over-reduction under stress conditions. For instance, it was shown that PTOX-dependent electron drainage accounted for up to 30% of total PSII electron flow in salt-stressed plants. PTOX is not bound to the thylakoid membrane in dark-adapted leaves but is associated with it at intense illumination and high transmembrane proton gradient (ΔpH) or membrane potential (Δψ). It was also shown that PTOX is capable of lateral translocation from stromal lamellae to granal thylakoid stacks under salt stress. Such changes in PTOX localization increase the accessibility of the substrate (plastoquinol) and the turnover rate of the enzyme. The available data allow considering PTOX as a possible target for manipulation to increase stress tolerance in sensitive plants.

Crucial role of the PTOX and CET pathways in optimizing ATP synthesis in mesophyll chloroplasts of C3 and C4 plants

Besides the linear electron flow (LET), several regulatory and alternative electron transfer pathways exist in chloroplasts, among them the cyclic electron transport around photosystem I (CET) and PTOX enzyme which oxidizes the plastoquinone pool. Until now has not been investigated the influence of PTOX activity on the regulation of CET pathways and hence the regulation of ATP production. The present study reveals differential responses of CET and PTOX regulation to environmental changes and how these pathways can adjust the ATP content to C3 and C4 metabolism. Here, we conducted a comparative study in maize (C4) and pea (C3) chloroplasts by measuring the activity of PSI in the presence of the inhibitors antimycin A and propyl gallate, estimating the content of thylakoid proteins that participate in CET, and determining ATP content and ATP synthase activity, and fluorescence analysis. We found that NDH-CET pathway plays an important role in maize chloroplasts, whereas PGR5/PGRL1 proteins play a dominant role in pea chloroplasts. The obtained results indicate a substantial involvement of PTOX activity in regulating electron flow in response to ATP demand. We also found that C3 and C4 chloroplasts contained quantitatively different CET membrane components. Based on the study findings, we propose a model of regulation of the linear electron transport (LET) and CET pathways in mesophyll chloroplasts of C4 and C3 plants under low light intensity based on ATP level and PTOX activity. • The NDH complex and PGR5/PGRL1 play a main role in CET in maize and pea mesophyll chloroplasts, respectively. • High PTOX activity in maize seems to be the key for producing ATP required for C4 metabolism. • PTOX is localized mainly in grana in maize and in stroma membranes in pea chloroplasts. • Antenna aggregation in maize indicates a high reduction, which may regulate ATP/NADPH demand.

The significance of WRKY45 transcription factor in metabolic adjustments during dark‐induced leaf senescence

Plants are constantly exposed to environmental changes that affect their performance. Metabolic adjustments are crucial to controlling energy homoeostasis and plant survival, particularly during stress. Under carbon starvation, coordinated reprogramming is initiated to adjust metabolic processes, which culminate in premature senescence. Notwithstanding, the regulatory networks that modulate transcriptional control during low energy remain poorly understood. Here, we show that the WRKY45 transcription factor is highly induced during both developmental and dark-induced senescence. The overexpression of Arabidopsis WRKY45 resulted in an early senescence phenotype characterized by a reduction of maximum photochemical efficiency of photosystem II and chlorophyll levels in the later stages of darkness. The detailed metabolic characterization showed significant changes in amino acids coupled with the accumulation of organic acids in WRKY45 overexpression lines during dark-induced senescence. Furthermore, the markedly upregulation of alternative oxidase (AOX1a, AOX1d) and electron transfer flavoprotein/ubiquinone oxidoreductase (ETFQO) genes suggested that WRKY45 is associated with a dysregulation of mitochondrial signalling and the activation of alternative respiration rather than amino acids catabolism regulation. Collectively our results provided evidence that WRKY45 is involved in the plant metabolic reprogramming following carbon starvation and highlight the potential role of WRKY45 in the modulation of mitochondrial signalling pathways.

An alkane monooxygenase (AlkB) family in which all electron transfer partners are covalently bound to the oxygen-activating hydroxylase

Alkane monooxygenase (AlkB) is a non-heme diiron enzyme that catalyzes the hydroxylation of alkanes. It is commonly found in alkanotrophic organisms that can live on alkanes as their sole source of carbon and energy. Activation of AlkB occurs via two-electron reduction of its diferric active site, which facilitates the binding, activation, and cleavage of molecular oxygen for insertion into an inert CH bond. Electrons are typically supplied by NADH via a rubredoxin reductase (AlkT) to a rubredoxin (AlkG) to AlkB, although alternative electron transfer partners have been observed. Here we report a family of AlkBs in which both electron transfer partners (a ferredoxin and a ferredoxin reductase) appear as an N-terminal gene fusion to the hydroxylase (ferr_ferrR_AlkB). This enzyme catalyzes the hydroxylation of medium chain alkanes (C6-C14), with a preference for C10-C12. It requires only NADH for activity. It is present in a number of bacteria that are known to be human pathogens. A survey of the genome neighborhoods in which is it found suggest it may be involved in alkane metabolism, perhaps facilitating growth of pathogens in non-host environments.

The Monoheme c Subunit of Respiratory Alternative Complex III Is Not Essential for Electron Transfer to Cytochrome aa3 in Flavobacterium johnsoniae.

ABSTRACTBacterial alternative complex III (ACIII) catalyzes menaquinol (MKH2) oxidation, presumably fulfilling the role of cytochromes bc1/b6f in organisms that lack these enzymes. The molecular mechanism of ACIII is unknown and so far the complex has remained inaccessible for genetic modifications. The recently solved cryo-electron microscopy (cryo-EM) structures of ACIII from Flavobacterium johnsoniae, Rhodothermus marinus, and Roseiflexus castenholzii revealed no structural similarity to cytochrome bc1/b6f and there were variations in the heme-containing subunits ActA and ActE. These data implicated intriguing alternative electron transfer paths connecting ACIII with its redox partner, and left the contributions of ActE and the terminal domain of ActA to the catalytic mechanism unclear. Here, we report genetic deletion and complementation of F. johnsoniae actA and actE and the functional implications of such modifications. Deletion of actA led to the loss of activity of cytochrome aa3 (a redox partner of ACIII in this bacterium), which confirmed that ACIII is the sole source of electrons for this complex. Deletion of actE did not impair the activity of cytochrome aa3, revealing that ActE is not required for electron transfer between ACIII and cytochrome aa3. Nevertheless, absence of ActE negatively impacted the cell growth rate, pointing toward another, yet unidentified, function of this subunit. Possible explanations for these observations, including a proposal of a split in electron paths at the ActA/ActE interface, are discussed. The described system for genetic manipulations in F. johnsoniae ACIII offers new tools for studying the molecular mechanism of operation of this enzyme.IMPORTANCE Energy conversion is a fundamental process of all organisms, realized by specialized protein complexes, one of which is alternative complex III (ACIII). ACIII is a functional analogue of well-known mitochondrial complex III, but operates according to a different, still unknown mechanism. To understand how ACIII interacts functionally with its protein partners, we developed a genetic system to mutate the Flavobacterium johnsoniae genes encoding ACIII subunits. Deletion and complementation of heme-containing subunits revealed that ACIII is the sole source of electrons for cytochrome aa3 and that one of the redox-active subunits (ActE) is dispensable for electron transfer between these complexes. This study sheds light on the operation of the supercomplex of ACIII and cytochrome aa3 and suggests a division in the electron path within ACIII. It also shows a way to manipulate protein expression levels for application in other members of the Bacteroidetes phylum.

Methylene blue treatment on Alzheimer and inflammatory bowel disease

High-efficiency oxidative phosphorylation plays a key role in the progression of many diseases. There is growing evidence of methylene blue’s protective and reversing actions against neurodegenerative and inflammatory bowel diseases through different mechanisms. In Alzheimer’s disease (AD), oligomeric amyloid beta accumulates in the mitochondria and contributes to mitochondrial dysfunction, which occurs before significant plaque deposition. Methylene blue provides an alternative mitochondrial electron transfer pathway, switching from high-efficiency oxidative phosphorylation to the low-efficiency aerobic glycolysis pathway by receiving electrons from NADH in the presence of complex I and transferring them to cytochrome C. The second mechanism is the inhibition of active caspases, especially Caspase-6, a cysteinyl protease causing inflammation and cell death, which has been associated with age-dependent cognitive decline and the pathology of sporadic and familial AD. The third mechanism is the reversal of tau aggregation by oxidizing cysteine residues in tau and forming a more stable monomer, thus blocking tau-tau bindings as well as clearing tau pathology through increased autophagy. In regards to inflammatory bowel disease, reducing oxidative stress and attenuating inflammatory pathways inhibits epithelial destruction in acetic acid-induced colitis. Methylene blue has an anti-colitis effect, mainly relying on its mitochondrial efficacy-restoring, antioxidative, anti-inflammatory, and anti-apoptotic properties. In summary, methylene blue is a promising agent for both AD and inflammatory bowel disease due to its beneficial effects as well as its low cost and high accessibility.

Metabolic strategies of dormancy of a marine bacterium Microbulbifer aggregans CCB-MM1: Its alternative electron transfer chain and sulfate-reducing pathway

Bacterial dormancy plays a crucial role in maintaining the functioning and diversity of microbial communities in natural environments. However, the metabolic regulations of the dormancy of bacteria in natural habitats, especially marine habitats, have remained largely unknown. A marine bacterium, Microbulbifer aggregans CCB-MM1 exhibits rod-to-coccus cell shape change during the dormant state. Therefore, to clarify the metabolic regulation of the dormancy, differential gene expression analysis based on RNA-Seq was performed between rod- (vegetative), intermediate, and coccus-shaped cells (dormancy). The RNA-Seq data revealed that one of two distinct electron transfer chains was upregulated in the dormancy. Dissimilatory sulfite reductase and soluble hydrogenase were also highly upregulated in the dormancy. In addition, induction of the dormancy of MM1 in the absence of MgSO4 was slower than that in the presence of MgSO4. These results indicate that the sulfate-reducing pathway plays an important role in entering the dormancy of MM1.

Mapping Influenza-Induced Posttranslational Modifications on Histones from CD8+ T Cells.

T cell function is determined by transcriptional networks that are regulated by epigenetic programming via posttranslational modifications (PTMs) to histone proteins and DNA. Bottom-up mass spectrometry (MS) can identify histone PTMs, whereas intact protein analysis by MS can detect species missed by bottom-up approaches. We used a novel approach of online two-dimensional liquid chromatography-tandem MS with high-resolution reversed-phase liquid chromatography (RPLC), alternating electron transfer dissociation (ETD) and collision-induced dissociation (CID) on precursor ions to maximize fragmentation of uniquely modified species. The first online RPLC separation sorted histone families, then RPLC or weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) separated species heavily clad in PTMs. Tentative identifications were assigned by matching proteoform masses to predicted theoretical masses that were verified with tandem MS. We used this innovative approach for histone-intact protein PTM mapping (HiPTMap) to identify and quantify proteoforms purified from CD8 T cells after in vivo influenza infection. Activation significantly altered PTMs following influenza infection, histone maps changed as T cells migrated to the site of infection, and T cells responding to secondary infections had significantly more transcription enhancing modifications. Thus, HiPTMap identified and quantified proteoforms and determined changes in CD8 T cell histone PTMs over the course of infection.

Photobactericidal activity activated by thiolated gold nanoclusters at low flux levels of white light

The emergence of antibiotic resistant bacteria is a major threat to the practice of modern medicine. Photobactericidal agents have obtained significant attention as promising candidates to kill bacteria, and they have been extensively studied. However, to obtain photobactericidal activity, an intense white light source or UV-activation is usually required. Here we report a photobactericidal polymer containing crystal violet (CV) and thiolated gold nanocluster ([Au25(Cys)18]) activated at a low flux levels of white light. It was shown that the polymer encapsulated with CV do not have photobactericidal activity under white light illumination of an average 312 lux. However, encapsulation of [Au25(Cys)18] and CV into the polymer activates potent photobactericidal activity. The study of the photobactericidal mechanism shows that additional encapsulation of [Au25(Cys)18] into the CV treated polymer promotes redox reactions through generation of alternative electron transfer pathways, while it reduces photochemical reaction type-ІІ pathways resulting in promotion of hydrogen peroxide (H2O2) production.

Metallated Schiff-Base Macromolecules as Alternative Metalloprotein Electron Transfer Intermediates

In the construction of biosensors, enzymes function as mediators converting biological signals generated by specific biological processes, into electrochemical signals. The ideology of bio-sensor design is retention of electron transfer activity of the enzyme utilizing superior interfacial architecture. In this work a Schiff-base macromolecule has been synthesized by reflux of 2, 3-diaminonaphthalene and pyrrole-2-carboxaldehyde starting materials. The Schiff-base ligand was subsequently complexed with FeCl2?4H2O under reflux, to produce the Fe-Schiff-base complex. The Schiff-base ligand and Fe-Schiff-base complex were characterized using nuclear magnetic resonance (NMR) spectroscopy, Ultra Violet/Visible (UV/Vis) spectroscopy, Fourier transfer infrared resonance (FTIR) and electron energy loss spectroscopy (EELS) to confirm the structure of the synthesis products. NMR spectroscopy confirmed the imide linkage of Schiff-base formation as two symmetrical peaks at 8.1 and 7.7 ppm respectively. Comparison of starting materials and product spectra by UV/Vis spectroscopy confirmed the disappearance of the diaminonaphthalene peak at 250 nm as evidence of complete conversion to product. FTIR spectroscopy of the Schiff-base ligand confirmed the formation of the imine bond at 1595 cm-1. EELS spectra comparing FeCl2?4H2O and the Fe-Schiff-base complex, showed good agreement in the energy loss profiles associated with changes to the electronic arrangement of Fe d-orbitals. EDS clearly identified a spectral band for Fe (7 - 8 eV) in the Fe-Schiff-base complex. Electrochemical evaluation of the Fe-Schiff-base complex was compared to the electrochemical signature of denatured cytochrome-C using cyclic voltammetry and square wave voltammetry. The Fe2+/Fe3+ quasi-reversible behavior for iron in the metallated complex was observed at -0.430 V vs. Ag/AgCl, which is consistent with reference values for iron in macromolecular structures.

Redirecting photosynthetic electron flux in the cyanobacterium Synechocystis sp. PCC 6803 by the deletion of flavodiiron protein Flv3

BackgroundOxygen-evolving photoautotrophic organisms, like cyanobacteria, protect their photosynthetic machinery by a number of regulatory mechanisms, including alternative electron transfer pathways. Despite the importance in modulating the electron flux distribution between the photosystems, alternative electron transfer routes may compete with the solar-driven production of CO2-derived target chemicals in biotechnological systems under development. This work focused on engineered cyanobacterial Synechocystis sp. PCC 6803 strains, to explore possibilities to rescue excited electrons that would normally be lost to molecular oxygen by an alternative acceptor flavodiiron protein Flv1/3—an enzyme that is natively associated with transfer of electrons from PSI to O2, as part of an acclimation strategy towards varying environmental conditions.ResultsThe effects of Flv1/3 inactivation by flv3 deletion were studied in respect to three alternative end-products, sucrose, polyhydroxybutyrate and glycogen, while the photosynthetic gas fluxes were monitored by Membrane Inlet Mass Spectrometry (MIMS) to acquire information on cellular carbon uptake, and the production and consumption of O2. The results demonstrated that a significant proportion of the excited electrons derived from photosynthetic water cleavage was lost to molecular oxygen via Flv1/3 in cells grown under high CO2, especially under high light intensities. In flv3 deletion strains these electrons could be re-routed to increase the relative metabolic flux towards the monitored target products, but the carbon distribution and the overall efficiency were determined by the light conditions and the genetic composition of the respective pathways. At the same time, the total photosynthetic capacity of the Δflv3 strains was systematically reduced, and accompanied by upregulation of oxidative glycolytic metabolism in respect to controls with the native Flv1/3 background.ConclusionsThe observed metabolic changes and respective production profiles were proposedly linked with the lack of Flv1/3-mediated electron transfer, and the associated decrease in the intracellular ATP/NADPH ratio, which is bound to affect the metabolic carbon partitioning in the flv3-deficient cells. While the deletion of flv3 could offer a strategy for enhancing the photosynthetic production of desired chemicals in cyanobacteria under specified conditions, the engineered target pathways have to be carefully selected to align with the intracellular redox balance of the cells.

Negative Ion Mode Collision-Induced Dissociation for Analysis of Protein Arginine Methylation

Arginine methylation is a common protein post-translational modification (PTM) that plays a key role in eukaryotic cells. Three distinct types of this modification are found in mammals: asymmetric Nη1Nη1-dimethylarginine (aDMA), symmetric Nη1Nη2-dimethylarginine (sDMA), and an intermediate Nη1-monomethylarginine (MMA). Elucidation of regulatory mechanisms of arginine methylation in living organisms requires precise information on both the type of the modified residues and their location inside the protein amino acid sequences. Despite mass spectrometry (MS) being the method of choice for analysis of multiple protein PTMs, unambiguous characterization of protein arginine methylation may not be always straightforward. Indeed, frequent internal basic residues of Arg methylated tryptic peptides hamper their sequencing under positive ion mode collision-induced dissociation (CID), the standardly used tandem mass spectrometry method, while the relative stability of the aDMA and sDMA side chains under alternative non-ergodic electron-based fragmentation techniques, electron-capture and electron transfer dissociations (ECD and ETD), may impede differentiation between the isobaric residues. Here, for the first time, we demonstrate the potential of the negative ion mode collision-induced dissociation MS for analysis of protein arginine methylation and present data revealing that the negative polarity approach can deliver both an unambiguous identification of the arginine methylation type and extensive information on the modified peptide sequences.

An alternative electron transfer process for selective detection of glucose in blood serum

Here, we demonstrate efficient electrochemical detection of glucose in blood serum using CdS nanoribbons coated indium Tin Oxide (ITO) electrode. Main appeal of this work is to establish an alternative electron transfer (AET) process by dissolving the glucose oxidase (GOX) enzyme in the electrolyte instead of direct immobilization on the electrode’s surface to demonstrate better performance of glucose sensing. In this context, we developed an experimental strategy through amperometric detection of hydrogen peroxide (H2O2) which is an intermediate reaction product of glucose oxidation by GOX present in the buffer solution. Therefore, we separately performed electrochemical detection of H2O2 by CdS nanoribbons in buffer solution followed by glucose detection with the same electrode by dissolving GOX in the buffer solution. The linear range of amperometric current measured with the same concentration of both H2O2 and glucose overlaps with each other which clearly proves that electrocatalytic reduction of both the analytes occur at the same rate on the CdS surface. Our studies reveal that dissolved GOX in buffer solution have a better performance than the GOX immobilized electrode. Furthermore, chronoamperometric detection of glucose in actual blood serum sample was performed and our results show even better performance than the commercially available glucometer.

Molecular Insights into Variable Electron Transfer in Amphibian Cryptochrome

Cryptochrome proteins are activated by the absorption of blue light, leading to the formation of radical pairs through electron transfer in the active site. Recent experimental studies have shown that once some of the amino acid residues in the active site of Xenopus laevis cryptochrome DASH are mutated, radical-pair formation is still observed. In this study, we computationally investigate electron-transfer pathways in the X. laevis cryptochrome DASH by extensively equilibrating a previously established homology model using molecular dynamics simulations and then mutating key amino acids involved in the electron transfer. The electron-transfer pathways are then probed by using tight-binding density-functional theory. We report the alternative electron-transfer pathways resolved at the molecular level and, through comparison of amino acid sequences for cryptochromes from different species, we demonstrate that one of these alternative electron-transfer pathways could be general for all cryptochrome DASH proteins.

Effect of the anode potential on the physiology and proteome of Shewanella oneidensis MR-1

Shewanella species respire using iron and manganese oxides as well as electrodes as solid terminal electron acceptors. Shewanella oneidenis MR-1 exploits mediated as well as direct extracellular electron transfer (EET) modes to transfer electrons at different formal potentials. These different EET modes at different potentials may utilise alternate electron transfer pathways. Therefore, we investigated how different anode potentials, providing different maximum microbial energy gains impacted S. oneidensis microbial physiology. Using quantitative proteomics, comparative analysis of the cellular variations to different anode potentials was performed. A label-free proteomic mass spectrometric analysis method, SWATH-MS, was used to gather quantitative information to determine physiological changes of Shewanella oneidensis MR-1 grown at different anodic potentials. S. oneidensis was cultured and grown in electrochemical cells at the set anode potentials of +0.71V, +0.21V & −0.19V versus SHE reference electrode, while the current production was monitored. At maximum current, electrodes were removed and whole-cell proteins extracted. Subsequent SWATH-MS analysis revealed information on 740 identified proteins across the three electrode potentials. For the first time, we show the abundance of S. oneidensis electron transfer proteins differs with electrode potential.

Alternative Electron-Transfer Channels Ensure Ultrafast Deactivation of Light-Induced Excited States in Riboflavin Binding Protein

Flavoproteins, containing flavin chromophores, are enzymes capable of transferring electrons at very high speeds. The ultrafast photoinduced electron-transfer (ET) kinetics of riboflavin binding protein to the excited riboflavin was studied by femtosecond spectroscopy and found to occur within a few hundred femtoseconds [ Zhong and Zewail, Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 11867-11872 ]. This ultrafast kinetics was attributed to the presence of two aromatic rings that could transfer the electron to riboflavin: the side chains of tryptophan 156 and tyrosine 75. However, the underlying ET mechanism remained unclear. Here, using a hybrid quantum mechanical-molecular dynamics approach, we perform ET dynamics simulations taking into account the motion of the protein and the solvent upon ET. This approach reveals that ET occurs via a major reaction channel involving tyrosine 75 (83%) and a minor one involving tryptophan 156 (17%). We also show that the protein environment is designed to ensure the fast quenching of the riboflavin excited state.

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP)

The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 107 M-1⋅s-1) and catalytically decomposes H2O2 using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km = 2.1 × 105 versus 3.5 × 104 M-1⋅s-1, respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H2O2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp233•+). Mutation of Trp233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp233•+, a Cys222-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H2O2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp233 and Cys222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.