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Geobacter sulfurreducens is of interest for the highest efficiency of power generation and extremely long extracellular electron transfer (EET) between the bacterium and electrodes. Despite more than 15 years of intensive molecular biological research, there is still no clear answer which molecules are responsible for these processes. In the present work, we look at the problem from another (atomic) perspective and identify the location and shape of the compounds that are known to be conductive, particularly those containing Fe atoms. By using highly sophisticated energy dispersive X-ray spectroscopy combined with high-angle annular dark-field transmission electron microscopy enabling detection, identification, and localization of chemical compounds on the surface at nearly atomic spatial resolution, we analyze Fe spatial distribution within the G. sulfurreducens community. We discover the presence of small Fe-containing particles on the surface of the bacterium cells. The size of the particles (diameter 5.6 nm) is highly reproducible and comparable with the size of a single protein. The particles cover about 2% of the cell surface, which is similar to that expected for molecular conductors responsible for electron transfer through the bacterium cell wall. We find that G. sulfurreducens filaments ("bacterial molecular wires") also contain Fe atoms in their bundles. We observe that the bacterium enable changing the distance between the Fe-containing bundles in the filaments from separated to attached (the latter is needed for the efficient electron transfer between the Fe-containing particles), depending on the bacterium metabolic activity and attachment to extracellular substrates. These results are consistent with the recently published research about the role of Fe atoms in protein molecular conductance ( Phys. Chem. Chem. Phys. , 2018 , 20 , 14072 - 14081 ) and show what type of Fe-containing particles are involved in the bacterial extracellular communication. They can be used for the design and construction of artificial biomolecular wires and bioinorganic interfaces.

The ability of certain microorganisms to live in a multi-cell thick, electrode-grown biofilm by utilizing the electrode as a metabolic electron acceptor or donor requires electron transfer across cell membranes, through the biofilm, and across the biofilm/electrode interface. Even for the most studied system, anode-grown Geobacter sulfurreducens, the mechanisms underpinning each process and how they connect is largely unresolved. Here we report on G. sulfurreducens biofilms grown across the gap separating two electrodes by maintaining one electrode at 0.300 V vs. Ag/AgCl (0.510 V vs. SHE) to act as a sustained metabolic electron acceptor while the second electrode was at open circuit. The poised electrode exhibited the characteristic current-time profile for electrode-dependent G. sulfurreducens biofilm growth. The open circuit potential (OCP) of the second electrode however increased after initially decreasing for 1.5–2 days. The increase in OCP is taken to indicate the point at which the growing biofilm bridged the gap between the electrodes, enabling cells in contact with the open circuit electrode to utilize the poised electrode as an electron acceptor. After but not prior to reaching this point, the second electrode was able to act as a sustainable electron acceptor immediately after being placed under potential control without requiring further time to develop. These results indicate that heterogeneous ET (H-ET) across the biofilm/electrode interface and long-distance ET (LD-ET) through the biofilm are highly correlated, if not inseparable, and may share many common components.

Protein molecular conductance has attracted attention from researchers for the possibility of constructing innovative flexible biocompatible nanoscale electronic devices and smart hybrid materials. Due to protein complexity, most evaluations of protein conductivity are based on the simple estimation of protein's molecular orbital energy levels and spatial distributions without analysing its protein interaction with electrodes and the calculation of the rates of electron transfer (ET). In the present work, we included in our density functional theory (DFT) analysis an approach based on the non-equilibrium Green's function (NEGF) allowing for calculation from the first principles the molecular interaction with electrodes and thus the role of electrode materials, Fermi level, the thermal distribution of electronic energy levels, and the coupling efficiency between the molecule and the electrodes. Compared to proteins studied so far, mainly artificial peptides, heme-containing cytochromes, and bacterial pili, we choose rubredoxin for our calculation. Rubredoxin contains a non-heme iron that, as we have discovered recently, can be involved in extracellular ET in electroactive bacterial biofilms (Yates et al., Energy Environ. Sci., 2016, 9, 3544–3558). Our calculations show that an iron atom incorporated into the protein structure as an iron–sulfur cluster opens a transmission path at the energy corresponding to the Fermi level of the electrodes. This allows the protein to become an extremely efficient conductor at very low bias voltages (

Fabrication of nanoscale structures with localized surface plasmons allows for substantial increase in sensitivity of chem/bio sensors. The main challenge for realizing complex nanoplasmonic structures in solution is the high level of precision required at the nanoscale to position metal nanoparticles in 3D. In this study, we report a virus-like particle (VLP) for building a 3D plasmonic nanostructure in solution in which gold nanoparticles are precisely positioned on the VLP by directed self-assembly techniques. These structures allow for concentration of electromagnetic fields in the desired locations between the gold nanoparticles or “hot spots”. We measure the efficiency of the optical field spatial concentration for the first time, which results in a ten-fold enhancement of the capsid Raman peaks. Our experimental results agree with our 3D finite element simulations. Furthermore, we demonstrate as a proof-of-principle that the plasmonic nanostructures can be utilized in DNA detection down to 0.25ng/μl (lowest concentration tested), while the protein peaks from the interior of the nanoplasmonic structures, potentially, can serve as an internal tracer for the biosensors.

Microbial electrosynthesis (ME) seeks to use electroautotrophy (the reduction of CO2 by microbial electrode catalysts) to generate useful multi-carbon compounds. It combines the utility of electrosynthesis with the durability of microorganisms and potential to engineer microbial metabolic processes. Central to achieving efficient ME is understanding the extracellular electron transport (EET) processes that enable certain microorganisms to utilize electrodes as metabolic electron donors. The Marinobacter-Chromatiaceae-Labrenzia (MCL) biocathode is an electroautotrophic biofilm-forming microbial community enriched from seawater that grows aerobically on gold or graphite cathodes, which we study to understand the mechanisms underpinning electroautotrophy. Evidence suggests that MCL reduces O2 using the cathode as its sole electron donor, directing a portion of the acquired electrons and energy to fix CO2 for biomass. A key feature of MCL is that it grows at +310 mV vs. SHE. Here, we apply electrochemical gating measurements, originally developed to study electron transport through polymer films, to study EET through living MCL biofilms. The results indicate that MCL biofilms employ a redox conduction mechanism to transport electrons across the biofilm/electrode interface and into the biofilm over multiple cell lengths (at least 5 μm) away from the electrode surface. In addition to making living MCL biofilms electrically conductive (60 μS cm−1 at 30 °C – more than 10 times greater conductivity than any other living microbial biofilm for which reliable measurements have been made), it enables electron uptake by cells not in direct contact with the electrode surface, which has not been previously reported for any biocathode. Confocal resonance Raman microscopy confirms the presence of c-type cytochromes as the putative redox cofactors involved in LD-EET, consistent with the activation energy for LD-EET obtained from the temperature dependency of the electrochemical gating measurements. These results provide the first report and mechanistic characterization of long-distance EET occurring within a multi-cell thick electroautotrophic biofilm – key milestones toward rational design and optimization of viable ME systems.

Here, we describe the long-distance (multi-cell-length) extracellular electron transport (LD-EET) that occurs in an anode-grown mixed community biofilm (MCB) enriched from river sediment that contains 3%-45% Geobacter spp. High signal-to-noise temperature-dependent electrochemical gating measurements (EGM) using interdigitated microelectrode arrays reveal a peak-shaped electrical conductivity vs. potential dependency, indicating MCB acts as a redox conductor, similar to pure culture anode-grown Geobacter sulfurreducens biofilms (GSB). EGM also reveal that the maximum sustained rate of LD-EET in MCB is comparable to GSB, and the same whether under acetate-oxidizing or acetate-free conditions. Voltammetry indicated that MCB possesses 3- to 5-fold less electrode-accessible redox cofactors than GSB, suggesting that MCB may be more efficiently organized than GSB for LD-EET or that a small portion of electrode accessible redox cofactors of GSB are involved in LD-EET. The activation energy for LD-EET (0.11 ± 0.01 eV) was comparable to GSB, consistent with the possible role of c-type cytochromes as LD-EET cofactors, detected in abundance by confocal resonance Raman microscopy. Taken together, the results demonstrate LD-EET for a mixed community anode-grown microbial biofilm that is remarkably similar to GSB even though it contains many different types of microorganisms and appears to utilize far fewer EET redox cofactors.

Geobacter sulfurreducens pili composed of the Type IV pili structural peptide PilA have been implicated as efficient electronic conductors. Though investigated experimentally, no detailed theoretical studies have been performed to date that provide quantitative estimation of the transmission spectrum, electron transfer (ET) paths, efficiency of current generation, and other factors needed for understanding possible mechanisms of conductivity. In the present work, we calculate from first principles the possibilities of electron tunneling through 3 PilA fragments which structure was identified recently by NMR. The results indicate that positively charged amino acids, arginines and lysines form electrostatic traps in the middle of the peptide preventing ET at low bias voltages (

When grown on the surface of an anode electrode, Geobacter sulfurreducens forms a multi-cell thick biofilm in which all cells appear to couple the oxidation of acetate with electron transport to the anode, which serves as the terminal metabolic electron acceptor. Just how electrons are transported through such a biofilm from cells to the underlying anode surface over distances that can exceed 20 microns remains unresolved. Current evidence suggests it may occur by electron hopping through a proposed network of redox cofactors composed of immobile outer membrane and/or extracellular multi-heme c-type cytochromes. In the present work, we perform a spatially resolved confocal resonant Raman (CRR) microscopic analysis to investigate anode-grown Geobacter biofilms. The results confirm the presence of an intra-biofilm redox gradient whereby the probability that a heme is in the reduced state increases with increasing distance from the anode surface. Such a gradient is required to drive electron transport toward the anode surface by electron hopping via cytochromes. The results also indicate that at open circuit, when electrons are expected to accumulate in redox cofactors involved in electron transport due to the inability of the anode to accept electrons, nearly all c-type cytochrome hemes detected in the biofilm are oxidized. The same outcome occurs when a comparable potential to that measured at open circuit (-0.30 V vs. SHE) is applied to the anode, whereas nearly all hemes are reduced when an exceedingly negative potential (-0.50 V vs. SHE) is applied to the anode. These results suggest that nearly all c-type cytochrome hemes detected in the biofilm can be electrochemically accessed by the electrode, but most have oxidation potentials too negative to transport electrons originating from acetate metabolism. The results also reveal a lateral heterogeneity (x-y dimensions) in the type of c-type cytochromes within the biofilm that may affect electron transport to the electrode.

Bacterial electroactive biofilms attract researchers’ attention due to their ability for generation of electric current when electrons travel for very long, up to several tens microns, distance. It was demonstrated that this electron transfer ability correlates with the accumulation in the BF a c-type cytochrome (Cyt), but molecular nature of this Cyt, its spatial localization, interaction with the environment, regulation and the controlling factors are not understood. Bacterial cell polarity is an internal cell asymmetry that correlates with the cell ability to sense energy and metabolite sources, chemical signals, quorum signals, toxins, and movement in the desired directions. This ability also plays central role in cell attachment to various surfaces and biofilm formation. In the present work, we develop a new experimental approach for analyzing redox potential spatial distribution within individual bacterial cells and use this approach for identification how attachment to inorganic mineral, mica affects redox state inside individual G. sulfurreducens cells. The approach is based on confocal Raman microscopy allowing for precise estimation of redox state and localization of subcellular cytochromes. Applying Gaussian deconvolution and principal component analysis we are able to identify spectral signatures of cytochromes in different redox states and intracellular locations. Our results give a new tool for quick identification of internal and surface exposed cytochromes and their localization at bacterial cell sides facing the substrate and the solution. They open up the possibility for analyzing electrochemical potentials inside individual bacterial cells and indicate that Cyt redox state might be a driving force switching cell metabolic activity that leads to irreversible cell attachment to electrode, cell-to-cell association, and biofilm formation. Nikolai Lebedev, Matthew Yates, Igor Griva, and Leonard M Tender: Internal Polarity of G. Sulfurreducens Bacterial Cells attached to an Inorganic Substrate. ChemPhysChem 19:1–11, 2018 Nikolai Lebedev, Igor Griva, Anders Blom and Leonard M. Tender Effect of iron doping on protein molecular conductance. - Physical Chemistry Chemical Physics 20: 14072-14081, 2018

Construction of efficient and highly integrated electronic devices is the main challenge and goal of nanoelectronics. The main problem in the construction of such devices is the fusion between conducting and controlling parts of the device, substantially reducing the efficiency of regulation. To approach the problem we study the electron transfer through a carbon nanotube (CNT)-histidine-heme-histidine-CNT conjugate with an orthogonal porphyrin orientation relative to the CNT electrodes. Using density functional theory and nonequilibrium Green’s function calculations we show that at low bias the CNT molecular orbitals are electronically coupled only to the Fe atom but uncoupled from the tetrapyrrole ring of the porphyrin. We found that at low bias the electrons pass exclusively through the central Fe atom of the porphyrin, but at higher bias they are partially scattered by the tetrapyrrole ring, leading to a reduction of the total current through the molecule (negative differential resistance). This allow...

Miniaturization of electronic devices to the level of single molecules requires detailed understanding of the mechanisms of their operation. One of the questions here is the identification of the role of structural alterations in charge separation and stabilization in photoactive complexes. To address this question, we calculate optimized molecular and electronic structures, and optical and vibrational spectra of l,l'-dimethyl 4,4'-bipyridinium-bis tetraphenylborate PQ(BPh(4))(2) complex ab initio using density functional theory approach and compare them with the experimentally observed UV-Vis and Raman spectra of the molecules in solid-state films. The results indicate that the association of PQ and BPh(4) leads to the formation of an internally ionized structure that is accompanied by the structural reorganization of both PQ (the twisting of pyridinium rings) and BPh(4) (phenyl rings rotation) moieties. The quanta of light do not seem to be directly involved in the formation of this ionized structure, but provide energy for fast recombination of the separated charges between BPh(4)(-) and PQ(2+). The high efficiency of the dark charge separation and the stabilization of separated charges in the complex permit the using of PQ(BPh(4))(2) in various charge-transfer devices like molecular probes, photovoltaic devices or chemical memory units.

Electron transfer (ET) through oxidized (QOPV) and reduced-protonated (HQOPV) forms of oligo(phenylene vinylene) quinone placed between two gold electrodes in the absence and presence of external water molecules is calculated using density functional theory with a nonequilibrium Green’s function method. The results show that the presence of hydrogen atoms as an internal component (in reduced-protonated form of the molecule) screens the stimulating effect of oxygen on QOPV conductivity and eliminates the efficiency of the molecule conductance at low bias voltages. The formation of a complex with water restores the HQOPV conductivity at low biases and opens several additional strong conductivity channels below the Fermi level, substantially improving the efficiency of ET between QOPV and the electrode, especially at longer distances between the molecule and the metal. This effect of water can be utilized in the construction of novel highly efficient electrochemically gated electronic devices. It also opens ...

Bacterial cell polarity is an internal asymmetric distribution of subcellular components, including proteins, lipids, and other molecules that correlates with the cell ability to sense energy and metabolite sources, chemical signals, quorum signals, toxins, and movement in the desired directions. This ability also plays central role in cell attachment to various surfaces and biofilm formation. Mechanisms and factors controlling formation of this cell internal asymmetry are not completely understood. As a step in this direction, in the present work, we develop an approach for analyzing how information about inorganic substrate can be non-genetically coded inside an individual bacterial cell. As a model system, we use G. sulfurreducens cells attached to an inorganic mineral, mica. The approach utilizes confocal Raman microscopy, Gaussian deconvolution, and Principal Component Analysis (PCA) and allows for quick label-free identification of the molecular signature of cytochrome intracellular location and the cell to substrate binding down to the level of individual bacterial cells. Our results describe a spectroscopic signature of cell adhesion and how the information about cell adhesion can be coded inside individual bacterial cells.

Electron transmission parameters of oligo(phenylene vinylene) quinone (OPVQ) placed between two gold electrodes were calculated ab initio using density functional theory (DFT) with the nonequilibri...

Bacterial electroactive biofilms attract researchers’ attention due to their ability for generation of electric current when electrons travel for very long, up to several tens microns, distance. It was demonstrated that this electron transfer (ET) ability correlates with the accumulation in the BF huge amounts of a c-type cytochrome (Cyt), but molecular nature of this Cyt, its spatial localization, interaction with the environment, regulation and the controlling factors are not completely understood. Bacterial cell polarity is an internal cell asymmetry that correlates with the cell ability to sense energy and metabolite sources, chemical signals, quorum signals, toxins, and movement in the desired directions. This ability also plays central role in cell attachment to various surfaces and biofilm formation. Biochemical analysis and molecular imaging demonstrate that sensing of energy and nutrients by the cell correlates with asymmetric protein, lipid and other molecule distributions within the cell. In the present work, we develop a new experimental approach for analyzing redox potential spatial distribution within individual bacterial cells and use this approach for identification how attachment to inorganic mineral, mica affects redox state inside individual G. sulfurreducens cells. The approach is based on confocal Raman microscopy allowing for precise estimation of redox state and localization of subcellular cytochromes. Applying Gaussian deconvolution and principal component analysis we are able to identify spectral signatures of cytochromes in different redox states and intracellular locations. Our results give a new tool for quick identification of internal and surface exposed cytochromes and their localization at bacterial cell sides facing the substrate and the solution. They open up the possibility for analyzing electrochemical potentials inside individual bacterial cells and indicate that Cyt redox state might be a driving force switching cell metabolic activity that leads to irreversible cell attachment to electrode, cell-to-cell association, and biofilm formation.

Specific and directed changes in structure between the bridge and headgroup can significantly change the rate for proton-coupled electron transfer of self-assembled monolayers containing quinone derivatives with varying electronic conjugation. We compared the apparent proton–coupled electron transfer rates at pHs between 1 and 12 of quinone self-assembled monolayers (SAMs) on gold electrodes in which the quinone compounds contained different bridging groups (i.e. a single, double or triple bond) connecting the headgroup to the oligo(phenylene vinylene) (OPV) thiol anchor. The quinone compounds in the SAMs were diluted by octane-1-thiol, and cyclic voltammograms of the quinone/hydroquinone (Q/H2Q) couple were measured as a function of scan rate. Using Laviron’s formalism, peak to peak separations of the anodic and cathodic peak potentials were analyzed to estimate apparent rate constants (kapp). We found that the mathematical expressions derived by Laviron for the two-electron, two-proton couple describing a nine member square-scheme can fit the data with an average single electron transfer rate constant reflecting the electron tunneling parameter, β, of the different bridging structures. In addition, several pKas involved in the nine member scheme shifted to higher values in the immobilized system when compared to reported values of hydroquinone in solution.

We describe a new strategy for the generation of self-assembled monolayers of three different quinones from derivatives each containing a short oligo(phenylene vinylene) (OPV) bridge with a methyl protected aryl thiol at the base and 2,5-dimethoxybenzene as the headgroup connected through either a single, double or triple bond. After partial deprotection of both −SCH 3 and −OCH 3 groups using t -BuSNa as a dealkylation agent, self-assembled monolayers (SAMs) of each compound were formed on gold. Complete deprotection of the headgroup to generate the hydroquinone/quinone redox couple was accomplished electrochemically in 0.1 M H 2 SO 4 using cyclic voltammetry. Electrochemical characterization as a function of the different bridging groups for the generated hydroquinone/quinone redox couple revealed formal potentials and redox reaction rates dramatically affected by small structural changes near the headgroup.

A homology model of NADPH:protochlorophyllide (Pchlide) oxidoreductase A (POR; E.C. 1.3.33.1) of barley is developed and verified by site-directed mutagenesis. PORA is considered a globular protein consisting of nine α-helices and seven β-strands. The model predicts the presence of two functionally distinctive Pchlide binding sites where the pigment is coordinated by cystein residues. The pigment bound to the first, high-affinity Pchlide binding site is used for the formation of the photoactive state of the enzyme. The pigment bound to the second, low-affinity Pchlide binding site is involved in the PORA:PORB interaction, allowing for resonance energy transfer between the neighboring PORs in the complex. In the in vitro reconstituted light-harvesting POR:Pchlide complex (LHPP), light absorbed by PORA-bound Pchlide b is transferred to PORB-bound Pchlide a . That induces the conversion of Pchlide a to chlorophyllide (Chlide) a . This energy transfer eliminates the possibility of Pchlide b photoreduction and prevents that excited triplet states of either Pchlides a or b accumulate and provoke singlet oxygen production. Together, our results provide a photoprotective role of PORA during greening.

We found that when a quinone headgroup, present in a mixed self-assembled monolayer on gold, reacts with a nucleophile, dissolved in the bulk phase, the reaction rate widely depends on the chemical nature of the tether, being 7 times faster for quinones attached via a delocalized bridge as compared to a saturated alkane chain. Cyclic voltammetry (CV) of the quinone/hydroquinone redox couple was used to monitor the nucleophilic addition, while simulated CVs compared to experimental runs permitted the determination of rate constants. Analysis of CV data also suggests that the delocalized oligo(phenylene ethynylene) bridge facilitates the addition of two mercaptoethanol molecules as compared to the alkane bridge, where only one molecule is being added. The use of delocalized bridges for tethering quinones to electrodes is of great potential in electrochemically controlled "tuning" of surfaces needed in biosensor applications.

The electron-transfer (ET) parameters for oriented and aligned monolayers of the bacterial photosynthetic reaction center (RC) from Rhodobacter sphaeroides formed on the top of self-assembled monolayers (SAMs) of alkanethiols of various lengths immobilized on gold electrode are estimated using cyclic voltammetry and photoelectrochemistry. Utilization of the unique polyHis tag in the protein and the Ni−NTA chelator complex in SAMs allows for specific protein orientation with the RC primary donor facing the electrode. To improve the efficiency of ET between the RC special pair and the electrode, an RC-Cyt complex was formed (J. Am. Chem. Soc. 2006, 128, 12044−12045). The results are analyzed in terms of integrated Marcus formalism, taking into account the density of electronic states in the metal. The dependence of the ET rate on the distance between RC and electrode demonstrates an adiabatic region up to 10 A, typical for other proteins, followed by a nonadiabatic area of electron tunneling with a β-factor...

We compare the electron transfer (ET) rates of hydroquinone self-assembled monolayers (SAMs) on gold electrodes containing either an alkane or pi-delocalized oligo(phenylene vinylene) (OPV) bridge at basic pH (between pH 8 and 12.6). The hydroquinone SAMs were diluted with octane-1-thiol. Cyclic voltammograms of the quinone/hydroquinone (Q/H 2 Q) couple allowed the peak-to-peak separation of the anodic and catholic peak potentials to be analyzed and apparent rate constants ( k app ) to be determined using Laviron’s formalism. We found that a simple rate law containing one equilibrium constant ( K AH ) and two rate constants ( k AH and k A ) describes the dependence of k app as a function of pH. From this model, the ratio of the rate constants for the protonated form H 2 Q, k AH (OPV) / k AH (alk) was ∼800, and the ratio of the de-protonated form, HQ − , k A(OPV) / k A(alk) was ∼2. The large ratio at pH K AH suggests that, for protonated H 2 Q, the ability of the OPV bridges to tunnel electrons significantly affects the overall rate. The small ratio at strongly basic pH (>p K AH ) implies that, for the de-protonated form HQ − , the overall ET rate is less sensitive to the bridge.

International audience; We present in this paper a new medium access control (MAC) scheme devoted to orthogonal frequency division multiple access (OFDMA) systems which aims at reducing collision probabilities during the channel request period. The proposed MAC relies on the classical carrier sense multiple access/collision avoidance (CSMA/CA) protocol with RTS / CTS ("Request To Send" / "Clear To Send") mechanism. The proposed method focus on the collision probability of RTS messages exploiting a multi-channel configuration for these messages while using the whole band for data transmissions. The protocol may be interpreted as an asynchronous frequency multiplexing of RTS messages. This method achieves strong performance gains in terms of throughput and latency especially in crowded networks. Index Terms—Carrier sense multiple access/collision avoidance (CSMA/CA), multiband, throughput, MAC protocol.

We describe the synthesis of two oligo(phenylene vinylene)s (OPVs) with a hydroquinone moiety and a thiol anchor group: 4-(2‘,5‘-dihydroxystyryl)benzyl thioacetate and 4-[4‘-(2‘ ‘,5‘ ‘-dihydroxystyryl)styryl]benzyl thioacetate. Monolayers on gold of these molecules were examined by electrochemical techniques to determine the electron transfer kinetics of the hydroquinone functionality (H2Q) through these delocalized tethers (“molecular wires”) as a function of pH. Between pH 4 and 9, rate constants were ca. 100-fold faster than for the same H2Q functionality confined to the surface via alkane tethers. Also, in this same pH range rate constants were independent of the length of the OPV bridge. These new electroactive molecules in which the hydroquinone functionality is wired to the gold surface by means of OPV tethers should be useful platforms for constructing bioelectronic devices such as biosensors, biofuel cells, and biophotovoltaic cells with a fast response time.

A new type of monolayer of photosynthetic reaction centers (RC) with the primary donor facing the carbon electrode has been constructed using a new bifunctional linker and genetically engineered protein. Comparison of protein in two different orientations with linkers binding to the opposite sides of the protein demonstrates the possibility of utilizing the constructed surfaces as photoelectronic devices. The results show improvement of the electron transfer efficiency when RC is bound with the primary donor (P) facing the electrode (P-side). In either protein orientation, electron transfer within the protein is unidirectional and when applying a voltage RC operates as a photorectifier. Electron transfer between the protein and carbon electrodes in the constructed devices is most likely occurring by tunneling.

Coupling of photosynthetic reaction centers (RCs) with inorganic surfaces is attractive for the identification of the mechanisms of interprotein electron transfer (ET) and for possible applications in construction of photo- and chemosensors. Here we show that RCs from Rhodobacter sphaeroides can be immobilized on gold surfaces with the RC primary donor looking towards the substrate by using a genetically engineered poly-histidine tag (His 7 ) at the C-terminal end of the M-subunit and a NiNTA terminated self-assembled monolayer (SAM). In the presence of an electron acceptor, ubiquinone-10, illumination of this RC electrode generates a cathodic photocurrent. The action spectrum of the photocurrent coincides with the absorption spectrum of RC and the photocurrent decreases in response to the herbicide, atrazine, confirming that the RC is the primary source of the photoresponse. Disruption of the NiNTARC bond by imidazole leads to about 80% reduction of the photocurrent indicating that most of the photoactive protein is specifically bound to the electrode through the linker.

Plants and photosynthetic bacteria contain protein−molecular complexes that harvest photons with nearly optimum quantum yield and an expected power conversion efficiency exceeding 20%. In this work, we demonstrate the integration of electrically active photosynthetic protein−molecular complexes in solid-state devices, realizing photodetectors and photovoltaic cells with internal quantum efficiencies of approximately 12%. Electronic integration of devices is achieved by self-assembling an oriented monolayer of photosynthetic complexes, stabilizing them with surfactant peptides, and then coating them with a protective organic semiconductor.

We report a rapid, simple, and sensitive assay that is potentially amenable to high throughput screening for analysis of 2,4,6-trinitrotoluene (TNT) present in aqueous solutions. The assay is based on the change in fluorescence emission intensity of a fluorescently labeled TNT analogue pre-bound to an anti-TNT antibody that occurs upon its competitive displacement by TNT. The assay can be performed in both cuvette- and 96-well plate-based formats. TNT at a level of 0.5 micro g L(-1) (0.5 ppb) was detected in phosphate buffered saline; detection improved to 0.05 micro g L(-1) (0.05 ppb) for TNT dissolved in artificial seawater.

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes hydrogen transfer from NADPH to protochlorophyllide (PChlide) in the course of chlorophyll biosynthesis in photosynthetic organisms and is involved in the regulation of the development of photosynthetic apparatus in higher plants, algae and cyanobacteria. To approach molecular factors determining the enzyme activity in a living cell, several mutants of POR from pea (Pisum sativum) with site-directed modifications in different parts of the enzyme were generated. The mutant enzymes were expressed in a R. capsulatus mutant deficient in BChl biosynthesis, and their catalytic activity and ability to integrate in bacterial metabolism were analyzed. Our results demonstrate that in heterologous bacterial cell system, higher plant POR is integrated in the porphyrin biosynthesis network and its activity leads to the formation of photosynthetic chlorophyll-proteins (CPs). The study of POR mutants in R. capsulatus reveals several POR domains important for the association of the enzyme with other subcellular components and for its catalytic activity, including identification of putative enzyme reaction center and substrate binding site. The study also demonstrated that an unknown structural factor is important for the formation of the enzyme photoactive complex in etiolated plants. Moreover, our findings suggest that POR might be directly involved in the regulation of the metabolism of other porphyrins.

Bacterial electroactive biofilms attract researchers’ attention due to their ability for generation of electric current when electrons travel for very long distance, up to several tens microns in the course of cell metabolic activity. It was demonstrated that this electron transfer (ET) ability correlates with the accumulation in the BF huge amounts of a c-type cytochrome (Cyt), but molecular nature of this Cyt, its spatial localization, interaction with the environment, regulation and the controlling factors are not completely understood. Raman spectro-electrochemical analysis is a very useful tool for nondestructive spatially-resolved in vivo study of ET mechanisms. But utilization and especially interpretation of Raman spectra requires deep understanding of their origin. Traditional approach to interpret Raman spectra is based on an assignment of specific Raman bands to the vibration of individual chemical bonds. Though some interesting results and conclusions were made using this approach, especially for small molecules, it does not allow for mapping Raman spectrum to complex organic and biological molecules like Cyt. Moreover, we and others have shown that most of Raman spectral bands of biomolecules are due to collective oscillation of several chemical bonds that requires another means of spectral description. This problem can be solved by computer simulation of Raman spectra allowing for identification of their structural origin and project/map them to molecular structure at sub molecular spatial resolution. This opens up the possibility of wise analysis of molecular interaction of individual biomolecules and their components. Spectroscopic analysis of more complex multi-structural and multicomponent biological systems, like biological cell and biofilms is often so complex that detection and separation of individual bands is very difficult to achieve. This problem can be solved by Principal Component Analysis (PCA) allowing for data squeezing to smaller number of the main, so called principle, components (PCs). Prescribing these PCs to specific classes of molecule allows for classification and comparative analysis of complex systems. In this presentation we will summarize our results on the application of Exploratory Confocal Raman Spectroscopic Analysis for identification of the main ET components (structure-functional relationship) in bacterial Raman spectra and organization of these ET components in multifunctional and multidimensional bacterial anodic and cathodic electroactive biofilms. We will show how interaction of the cells with inorganic substrates affects their metabolic activity; how cells talk to each other about that; and how electron transfer through an individual bacterial cell can be monitored.

In this paper we provide an analytical expression of the capacity outage probability for Coordinated Multiple Point (CoMP) downlink transmission in Rayleigh fading. The theoretical derivation and simulation results match perfectly. Used in dense urban interference limited networks, CoMP is expected to improve cell-edge users' throughput and allow a better coverage and fairness. We demonstrate that our closed-form derivation can be helpful to maximise the capacity with outage and the goodput, by tuning appropriately the transmission data-rate and selecting optimal set of cooperating stations.

Metagenomic and metaproteomic analyses were utilized to determine the composition and function of complex air–water interface biofilms sampled from the hulls of two US Navy destroyers. Prokaryotic community analyses using PhyloChip-based 16S rDNA profiling revealed two significantly different and taxonomically rich biofilm communities (6,942 taxa) in which the majority of unique taxa were ascribed to members of the Gammaproteobacteria, Alphaproteobacteria and Clostridia. Although metagenomic sequencing indicated that both biofilms were dominated by prokaryotic sequence reads (> 91%) with the majority of the bacterial reads belonging to the Alphaproteobacteria, the Ship-1 metagenome harbored greater organismal and functional diversity and was comparatively enriched for sequences from Cyanobacteria, Bacteroidetes and macroscopic eukaryotes, whereas the Ship-2 metagenome was enriched for sequences from Proteobacteria and microscopic photosynthetic eukaryotes. Qualitative liquid chromatography-tandem mass spectrometry metaproteome analyses identified 678 unique proteins, revealed little overlap in species and protein composition between the ships and contrasted with the metagenomic data in that ~80% of classified and annotated proteins were of eukaryotic origin and dominated by members of the Bacillariophyta, Cnidaria, Chordata and Arthropoda (data deposited to the ProteomeXchange, identifier PXD000961). Within the shared metaproteome, quantitative 18O and iTRAQ analyses demonstrated a significantly greater abundance of structural proteins from macroscopic eukaryotes on Ship-1 and diatom photosynthesis proteins on Ship-2. Photosynthetic pigment composition and elemental analyses confirmed that both biofilms were dominated by phototrophic processes. These data begin to provide a better understanding of the complex organismal and biomolecular composition of marine biofilms while highlighting caveats in the interpretation of stand-alone environmental ‘-omics’ datasets.

International audience; —In this work we study the collision probability, saturation throughput and statistical delay for the carrier sense multiple access collision avoidance (CSMA/CA) protocol with request to send and clear to send (RTS/CTS) mechanism in the case of frequency channel division. We propose in this paper a modified version of CSMA/CA-RTS/CTS to be compatible with the channel repartition technique and we prove that an important gain is introduced in terms of system performance especially for loaded networks. Simulations highlight that dividing the channel into independent sub-channels reduces drastically the RTS collision probability. Moreover, a gain in terms of saturation throughput and delay is shown especially in dense networks. Index Terms—Carrier senses multiple access/collision avoid-ance (CSMA/CA), Frequency channel division, RTS/CTS, MAC protocol.

Atomic force microscopy and confocal resonance Raman microscopy (CRRM) of single-cells were used to study the transition of anode-grown Geobacter sulfurreducens biofilms from lag phase (initial period of low current) to exponential phase (subsequent period of rapidly increasing current). Results reveal that lag phase biofilms consist of lone cells and tightly packed single-cell thick clusters crisscrossed with extracellular linear structures that appears to be comprised of nodules approximately 20 nm in diameter aligned end to end. By early exponential phase, cell clusters expand laterally and a second layer of closely packed cells begins to form on top of the first. Abundance of c-type cytochromes (c-Cyt) is threefold greater in two-cell thick regions than in one-cell thick regions. The results indicate that early biofilm growth involves two transformations. The first is from lone cells to two-dimensionally associated cells during lag phase when current remains low. This is accompanied by formation of extracellular linear structures. The second is from two- to three-dimensionally associated cells during early exponential phase when current begins to increase rapidly. This is accompanied by a dramatic increase in c-Cyt abundance.

The mechanism of the protochlorophyllide (PChlide) photoreduction reaction operating in light-adapted plants and catalyzed by NADPH:protochlorophyllide oxidoreductase B (PORb) has been analyzed by low-temperature fluorescence spectroscopy by using purified barley PORb overexpressed heterologously in Escherichia coli as a fusion protein with the maltose-binding protein. We show that the PORb-catalyzed PChlide reduction reaction consists of two steps, one photochemical and the other nonphotochemical. The initial photochemical reaction follows a single quantum mechanism and leads to the formation of an unstable intermediate with mixed pigment electronic structure and an EPR spectrum that suggests the presence of a free electron. The second step involves the spontaneous conversion of the unstable intermediate into chlorophyllide as defined by its spectroscopic characteristics and migration on an HPLC column. Both steps of the reaction can be performed at subzero temperatures in frozen samples, suggesting that they do not include major changes in enzyme conformation or pigment rearrangement within the active site. The rate of the reaction at room temperature depends linearly on enzyme and substrate (PChlide) concentration, and the kinetic parameters are consistent with one molecule of substrate bound per active monomer in solution. The PORb-catalyzed reaction in vitro is spectroscopically similar to that identified in leaves of light-adapted plants, suggesting that the same reaction sequence observed operates in planta .

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide (pchlide) to chlorophyllide (chlide) in the biosynthesis of chlorophyll. POR is a peripheral membrane protein that accumulates to high levels in the prolamellar bodies of vascular plant etioplasts and is present at low levels in the thylakoid membranes of developing and mature plastids. Clustered charged-to-alanine scanning mutagenesis of the pea (Pisum sativum L.) POR was carried out and the resulting mutant enzymes analyzed for their ability to catalyze pchlide photoconversion in vivo and to associate properly with thylakoid membrane preparations in vitro. Of 37 mutant enzymes examined, 5 retained wild-type levels of activity, 14 were catalytically inactive, and the remaining 18 exhibited altered levels of function. Several of the mutant enzymes showed temperature- dependent enzymatic activity, being inactive at 32 °C, but partially active at 24 °C. Mutations in predicted α- helical regions of the protein showed the least effect on enzyme activity, whereas mutations in predicted β-sheet regions of the protein showed a consistent adverse affect on enzyme function. In the absence of added NADPH, neither wild-type POR nor any of the mutant PORs resisted proteolysis by thermolysin following assembly onto the thylakoid membranes. In contrast, when NADPH was present in the assay mixture, 13 of the 37 mutant PORs examined were found to be resistant to thermolysin upon treatment, suggesting that the mutations did not affect their ability to be properly attached to the thylakoid membrane. In general, the replacement of charged amino acids by alanine in the most N- and C-terminal regions of the mature protein did not significantly affect POR assembly, whereas mutations within the central core of the protein (between residues 86 and 342) were incapable of proper attachment to the thylakoid. Failure to properly associate with the thylakoid membrane in a protease resistant manner was only weakly correlated to loss of catalytic function. These studies are a first step towards defining structural determinants crucial to POR function and intraorganellar localization.