Nikolai Lebedev

Electrochemically controlled conductance switching in a single molecule: quinone-modified oligo(phenylene vinylene).

Reversible conductance switching in single quinone-oligo(phenylene vinylene) (Q-OPV) molecules was demonstrated using electrochemical STM. The switching was achieved by application of electrochemical potential to the substrate supporting the molecule. The ratio of conductances between the high- and low-conductivity states is over 40. The high-conductivity state is ascribed to strong electron delocalization of the fully conjugated hydroquinone-OPV structure, whereas the low-conductivity state is characterized by disruption of electron delocalization in the quinone-OPV structure.

Increasing Efficiency of Photoelectronic Conversion by Encapsulation of Photosynthetic Reaction Center Proteins in Arrayed Carbon Nanotube Electrode

The construction of efficient light energy converting (photovoltaic and photoelectronic) devices is a current and great challenge in science and technology and one that will have important economic consequences. Here we show that the efficiency of these devices can be improved by the utilization of a new type of nano-organized material having photosynthetic reaction center proteins encapsulated inside carbon nanotube arrayed electrodes. In this work, a generically engineered bacterial photosynthetic reaction center protein with specifically synthesized organic molecular linkers were encapsulated inside carbon nanotubes and bound to the inner tube walls in unidirectional orientation. The results show that the photosynthetic proteins encapsulated inside carbon nanotubes are photochemically active and exhibit considerable improvement in the rate of electron transfer and the photocurrent density compared to the material constructed from the same components in traditional lamella configuration.

Spatially Resolved Confocal Resonant Raman Microscopic Analysis of Anode-Grown Geobacter sulfurreducens Biofilms

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.

Adaptation of the Black Yeast Wangiella dermatitidis to Ionizing Radiation: Molecular and Cellular Mechanisms

Observations of enhanced growth of melanized fungi under low-dose ionizing radiation in the laboratory and in the damaged Chernobyl nuclear reactor suggest they have adapted the ability to survive or even benefit from exposure to ionizing radiation. However, the cellular and molecular mechanism of fungal responses to such radiation remains poorly understood. Using the black yeast Wangiella dermatitidis as a model, we confirmed that ionizing radiation enhanced cell growth by increasing cell division and cell size. Using RNA-seq technology, we compared the transcriptomic profiles of the wild type and the melanin-deficient wdpks1 mutant under irradiation and non-irradiation conditions. It was found that more than 3000 genes were differentially expressed when these two strains were constantly exposed to a low dose of ionizing radiation and that half were regulated at least two fold in either direction. Functional analysis indicated that many genes for amino acid and carbohydrate metabolism and cell cycle progression were down-regulated and that a number of antioxidant genes and genes affecting membrane fluidity were up-regulated in both irradiated strains. However, the expression of ribosomal biogenesis genes was significantly up-regulated in the irradiated wild-type strain but not in the irradiated wdpks1 mutant, implying that melanin might help to contribute radiation energy for protein translation. Furthermore, we demonstrated that long-term exposure to low doses of radiation significantly increased survivability of both the wild-type and the wdpks1 mutant, which was correlated with reduced levels of reactive oxygen species (ROS), increased production of carotenoid and induced expression of genes encoding translesion DNA synthesis. Our results represent the first functional genomic study of how melanized fungal cells respond to low dose ionizing radiation and provide clues for the identification of biological processes, molecular pathways and individual genes regulated by radiation.

Function and Regulation of Vibrio campbellii Proteorhodopsin: Acquired Phototrophy in a Classical Organoheterotroph

Proteorhodopsins (PRs) are retinal-binding photoproteins that mediate light-driven proton translocation across prokaryotic cell membranes. Despite their abundance, wide distribution and contribution to the bioenergy budget of the marine photic zone, an understanding of PR function and physiological significance in situ has been hampered as the vast majority of PRs studied to date are from unculturable bacteria or culturable species that lack the tools for genetic manipulation. In this study, we describe the presence and function of a horizontally acquired PR and retinal biosynthesis gene cluster in the culturable and genetically tractable bioluminescent marine bacterium Vibrio campbellii. Pigmentation analysis, absorption spectroscopy and photoinduction assays using a heterologous over-expression system established the V. campbellii PR as a functional green light absorbing proton pump. In situ analyses comparing PR expression and function in wild type (WT) V. campbellii with an isogenic ΔpR deletion mutant revealed a marked absence of PR membrane localization, pigmentation and light-induced proton pumping in the ΔpR mutant. Comparative photoinduction assays demonstrated the distinct upregulation of pR expression in the presence of light and PR-mediated photophosphorylation in WT cells that resulted in the enhancement of cellular survival during respiratory stress. In addition, we demonstrate that the master regulator of adaptive stress response and stationary phase, RpoS1, positively regulates pR expression and PR holoprotein pigmentation. Taken together, the results demonstrate facultative phototrophy in a classical marine organoheterotrophic Vibrio species and provide a salient example of how this organism has exploited lateral gene transfer to further its adaptation to the photic zone.

Toward understanding long-distance extracellular electron transport in an electroautotrophic microbial community

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.

Proteomic Analysis and Identification of the Structural and Regulatory Proteins of the Rhodobacter capsulatus Gene Transfer Agent

The gene transfer agent of Rhodobacter capsulatus (GTA) is a unique phage-like particle that exchanges genetic information between members of this same species of bacterium. Besides being an excellent tool for genetic mapping, the GTA has a number of advantages for biotechnological and nanoengineering purposes. To facilitate the GTA purification and identify the proteins involved in GTA expression, assembly and regulation, in the present work we construct and transform into R. capsulatus Y262 a gene coding for a C-terminally His-tagged capsid protein. The constructed protein was expressed in the cells, assembled into chimeric GTA particles inside the cells and excreted from the cells into surrounding medium. Transmission electron micrographs of phosphotungstate-stained, NiNTA-purified chimeric GTA confirm that its structure is similar to normal GTA particles, with many particles composed both of a head and a tail. The mass spectrometric proteomic analysis of polypeptides present in the GTA recovered outside the cells shows that GTA is composed of at least 9 proteins represented in the GTA gene cluster including proteins coded for by Orfs 3, 5, 6-9, 11, 13, and 15.

High Resolution AFM and Single-Cell Resonance Raman Spectroscopy of Geobacter sulfurreducens Biofilms Early in Growth

AFM 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 appear 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 > 3-fold greater in 2-cell thick regions than in 1-cell thick regions. The results indicate that early biofilm growth involves two transformations. The first is from lone cells to 2-dimensionally associated cells during lag phase when current remains low. This is accompanied by formation of extracellular linear structures. The second is from 2- to 3-dimensionally associated cells during early exponential phase when current begins to increases rapidly. This is accompanied by a dramatic increase in c-Cyt abundance.

POR structural domains important for the enzyme activity in R. capsulatus complementation system

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.

Structural Reorganizations Control Intermolecular Conductance and Charge Trapping in Paraquat-Tetraphenylborate Inverse Photochemical Cell

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(BPh4)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 BPh4 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 BPh4 (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 BPh4− and PQ2+. The high efficiency of the dark charge separation and the stabilization of separated charges in the complex permit the using of PQ(BPh4)2 in various charge‐transfer devices like molecular probes, photovoltaic devices or chemical memory units.