Midpoint Potential Research Articles

Cytochrome OmcZ is essential for the current generation by Geobacter sulfurreducens under low electrode potential

Geobacter sulfurreducens is a model exoelectrogen known for its capability to generate high current density. Microbes genetically related to G. sulfurreducens often dominate the anodic microbial consortia in the microbial electrochemical systems. Electroanalyses had revealed that G. sulfurreducens utilizes at least three electron transfer (ET) pathways. The ET pathway with a mid-point potential of − 0.22V is responsible for the reduction of low-potential electron acceptors. Nonetheless, the identity of the corresponding ET proteins remained unknown. This study compared the voltammograms of wild type G. sulfurreducens to those of a strain deficient in the cytochrome OmcZ (strain ΔomcZ). In bioelectrochemical reactors equipped with Indium tin oxide glasses as the working electrodes, the ΔomcZ strain generated lower current density (28.1±8.2μA/cm2) compared to that of the wild strain (82.9±16.7μA/cm2). For ΔomcZ, the catalytic wave in the low-potential region disappeared from the biofilm voltammogram. Differential pulse voltammetries also demonstrated the loss of a low-potential redox couple. OmcZ is therefore likely to be part of the low-potential pathway. Nevertheless, pathways with higher mid-point potentials remained functional after the deletion of the omcZ gene. OmcZ is hence probably specifically required for the low-potential ET pathway. On the other hand, mutant strain deficient in the omcB gene, which was previously found to be up-regulated under current-generating conditions, exhibited similar voltammetric patterns to the wild strain.

Cellular Assays for Ferredoxins: A Strategy for Understanding Electron Flow through Protein Carriers That Link Metabolic Pathways.

The ferredoxin (Fd) protein family is a structurally diverse group of iron-sulfur proteins that function as electron carriers, linking biochemical pathways important for energy transduction, nutrient assimilation, and primary metabolism. While considerable biochemical information about individual Fd protein electron carriers and their reactions has been acquired, we cannot yet anticipate the proportion of electrons shuttled between different Fd-partner proteins within cells using biochemical parameters that govern electron flow, such as holo-Fd concentration, midpoint potential (driving force), molecular interactions (affinity and kinetics), conformational changes (allostery), and off-pathway electron leakage (chemical oxidation). Herein, we describe functional and structural gaps in our Fd knowledge within the context of a sequence similarity network and phylogenetic tree, and we propose a strategy for improving our understanding of Fd sequence-function relationships. We suggest comparing the functions of divergent Fds within cells whose growth, or other measurable output, requires electron transfer between defined electron donor and acceptor proteins. By comparing Fd-mediated electron transfer with biochemical parameters that govern electron flow, we posit that models that anticipate energy flow across Fd interactomes can be built. This approach is expected to transform our ability to anticipate Fd control over electron flow in cellular settings, an obstacle to the construction of synthetic electron transfer pathways and rational optimization of existing energy-conserving pathways.

Anode potential influences the structure and function of anodic electrode and electrolyte-associated microbiomes.

Three bioelectrochemical systems were operated with set anode potentials of +300 mV, +550 mV and +800 mV vs. Standard Hydrogen Electrode (SHE) to test the hypothesis that anode potential influences microbial diversity and is positively associated with microbial biomass and activity. Bacterial and archaeal diversity was characterized using 16 S rRNA gene amplicon sequencing, and biofilm thickness was measured as a proxy for biomass. Current production and substrate utilization patterns were used as measures of microbial activity and the mid-point potentials of putative terminal oxidases were assessed using cyclic voltammetry. All measurements were performed after 4, 16, 23, 30 and 38 days. Microbial biomass and activity differed significantly between anode potentials and were lower at the highest potential. Anodic electrode and electrolyte associated community composition was also significantly influenced by anode potential. While biofilms at +800 mV were thinner, transferred less charge and oxidized less substrate than those at lower potentials, they were also associated with putative terminal oxidases with higher mid-point potentials and generated more biomass per unit charge. This indicates that microbes at +800 mV were unable to capitalize on the potential for additional energy gain due to a lack of adaptive traits to high potential solid electron acceptors and/or sensitivity to oxidative stress.

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD: A TOOL FOR REDOX REGULATION IN AN ANAEROBE

A recent report suggested that the thioredoxin-dependent metabolic regulation, which is widespread in all domains of life, existed in methanogenic archaea about 3.5 billion years ago. We now show that the respective electron delivery enzyme (thioredoxin reductase, TrxR), although structurally similar to flavin-containing NADPH-dependent TrxRs (NTR), lacked an NADPH-binding site and was dependent on reduced coenzyme F420 (F420H2), a stronger reductant with a mid-point redox potential (E'0) of -360 mV; E'0 of NAD(P)H is -320 mV. Because F420 is a deazaflavin, this enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). It transferred electrons from F420H2 to thioredoxin via protein-bound flavin; Km values for thioredoxin and F420H2 were 6.3 and 28.6 μm, respectively. The E'0 of DFTR-bound flavin was approximately -389 mV, making electron transfer from NAD(P)H or F420H2 to flavin endergonic. However, under high partial pressures of hydrogen prevailing on early Earth and present day deep-sea volcanoes, the potential for the F420/F420H2 pair could be as low as -425 mV, making DFTR efficient. The presence of DFTR exclusively in ancient methanogens and mostly in the early Earth environment of deep-sea volcanoes and DFTR's characteristics suggest that the enzyme developed on early Earth and gave rise to NTR. A phylogenetic analysis revealed six more novel-type TrxR groups and suggested that the broader flavin-containing disulfide oxidoreductase family is more diverse than previously considered. The unprecedented structural similarities between an F420-dependent enzyme (DFTR) and an NADPH-dependent enzyme (NTR) brought new thoughts to investigations on F420 systems involved in microbial pathogenesis and antibiotic production.

The unusual redox properties of C-type oxidases

Cytochrome cbb3 (also known as C-type) oxidases belong to the family of heme-copper terminal oxidases which couple at the end of the respiratory chain the reduction of molecular oxygen into water and the pumping of protons across the membrane. They are expressed most often at low pressure of O2 and they exhibit a low homology of sequence with the cytochrome aa3 (A-type) oxidases found in mitochondria. Their binuclear active site comprises a high-spin heme b3 associated with a CuB center. The protein also contains one low-spin heme b and 3 hemes c. We address here the redox properties of cbb3 oxidases from three organisms, Rhodobacter sphaeroides, Vibrio cholerae and Pseudomonas stutzeri by means of electrochemical and spectroscopic techniques. We show that the redox potential of the heme b3 exhibits a relatively low midpoint potential, as in related cytochrome c-dependent nitric oxide reductases. Potential implications for the coupled electron transfer and proton uptake mechanism of C-type oxidases are discussed.

The Heme-Based Oxygen Sensor Rhizobium etli FixL: Influence of Auxiliary Ligands on Heme Redox Potential and Implications on the Enzyme Activity

Conformational changes associated to sensing mechanisms of heme-based protein sensors are a key molecular event that seems to modulate not only the protein activity but also the potential of the FeIII/II redox couple of the heme domain. In this work, midpoint potentials (Em) assigned to the FeIII/II redox couple of the heme domain of FixL from Rhizobium etli (ReFixL) in the unliganded and liganded states were determined by spectroelectrochemistry in the presence of inorganic mediators. In comparison to the unliganded ReFixL protein (+19mV), the binding to ligands that switch off the kinase activity induces a negative shift, i. e. Em=−51, −57 and −156mV for O2, imidazole and CN−, respectively. Upon binding to CO, which does not affect the kinase active, Em was observed at +21mV. The potential values observed for FeIII/II of the heme domain of ReFixL upon binding to CO and O2 do not follow the expected trend based on thermodynamics, assuming that positive potential shift would be expected for ligands that bind to and therefore stabilize the FeII state. Our results suggest that the conformational changes that switch off kinase activity upon O2 binding have knock-on effects to the local environment of the heme, such as solvent rearrangement, destabilize the FeII state and counterbalances the FeII-stabilizing influence of the O2 ligand.

Yeast surface display of dehydrogenases in microbial fuel-cells

Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of −28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm−2 using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm−2 using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems.

Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase.

Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially valuable compounds. In this report, full length CYP79A1, CYP71E1 and POR of the dhurrin pathway in Sorghum bicolor were reconstituted individually in nanoscale lipid patches, “nanodiscs”, and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 revealed reversible redox peaks with average midpoint potentials of 80 ± 5 mV and 72 ± 5 mV vs. Ag/AgCl, respectively. POR yielded two pairs of redox peaks with midpoint potentials of 90 ± 5 mV and −300 ± 10 mV, respectively. The average heterogeneous electron transfer rate constant was calculated to be ~1.5 s−1. POR was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions. It is also a prelude for driving plant P450 systems electronically for simplified and cost-effective screening of potential substrates/inhibitors and fabrication of nano-bioreactors for synthesis of high value natural products.

From low- to high-potential bioenergetic chains: Thermodynamic constraints of Q-cycle function

The electrochemical parameters of all cofactors in the supercomplex formed by the Rieske/cytb complex and the SoxM/A-type O2-reductase from the menaquinone-containing Firmicute Geobacillus stearothermophilus were determined by spectroelectrochemistry and EPR redox titrations. All redox midpoint potentials (Em) were found to be lower than those of ubi- or plastoquinone-containing systems by a value comparable to the redox potential difference between the respective quinones. In particular, Em values of +200mV, −360mV, −220mV and −50mV (at pH7) were obtained for the Rieske cluster, heme bL, heme bH and heme ci, respectively. Comparable values of −330mV, −200mV and +120mV for hemes bL, bH and the Rieske cluster were determined for an anaerobic Firmicute, Heliobacterium modesticaldum. Thermodynamic constraints, optimization of proton motive force build-up and the necessity of ROS-avoidance imposed by the rise in atmospheric O2 2.5billionyears ago are discussed as putative evolutionary driving forces resulting in the observed redox upshift. The close conservation of the entire redox landscape between low and high potential systems suggests that operation of the Q-cycle requires the precise electrochemical tuning of enzyme cofactors to the quinone substrate as stipulated in P. Mitchell's hypothesis.

A Redox Active [2Fe-2S] Cluster on the Hydrogenase Maturase HydF.

[FeFe]-hydrogenases are nature's most prolific hydrogen catalysts, excelling at facilely interconverting H2 and protons. The catalytic core common to all [FeFe]-hydrogenases is a complex metallocofactor, referred to as the H-cluster, which is composed of a standard [4Fe-4S] cluster linked through a bridging thiolate to a 2Fe subcluster harboring dithiomethylamine, carbon monoxide, and cyanide ligands. This 2Fe subcluster is synthesized and inserted into [FeFe]-hydrogenase by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG are radical S-adenosylmethionine enzymes and synthesize the nonprotein ligands of the H-cluster. HydF is a GTPase that functions as a scaffold or carrier for 2Fe subcluster production. Herein, we utilize UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopic studies to establish the existence of redox active [4Fe-4S] and [2Fe-2S] clusters bound to HydF. We have used spectroelectrochemical titrations to assign iron-sulfur cluster midpoint potentials, have shown that HydF purifies with a reduced [2Fe-2S] cluster in the absence of exogenous reducing agents, and have tracked iron-sulfur cluster spectroscopic changes with quaternary structural perturbations. Our results provide an important foundation for understanding the maturation process by defining the iron-sulfur cluster content of HydF prior to its interaction with HydE and HydG. We speculate that the [2Fe-2S] cluster of HydF either acts as a placeholder for HydG-derived Fe(CO)2CN species or serves as a scaffold for 2Fe subcluster assembly.

Calculations of the Energetics of Oxidation of Aqueous Nucleosides and the Effects of Prototropic Equilibria.

Recently the calculated standard reduction potentials of the radical-cations of N-methyl substituted DNA bases have been reported that agree fairly well with the experimental results. However, there are issues reflecting the fact that the experimental results usually relate to the couple E(o)(Nuc(•),H(+)/NucH(+)), whereas the calculated results are for the E(o)(Nuc(•+)/Nuc) couple. To calculate the midpoint reduction potential at pH 7 (Em7), it is important to have accurate acid dissociation constants (pKs) for both ground-state bases and their radicals, and the effects of uncertainty in some of these values (e.g., that of the adenosine radical) must be considered. Calculations of the pKs of the radicals of the nucleic acid bases (as nucleosides) have been performed to explore the effects the various pKs have on calculating the values of Em7 and to see what improvements can be made with the accuracy of the calculations.

Carbon paper electrode modified with TiO2 nanowires enhancement bioelectricity generation in microbial fuel cell

A novel TiO2 nanowires (TiO2 -NWs) successfully modified a carbon paper (CP) substrate through hydrothermal reaction was used as bioanode in microbial fuel cell (MFC). TiO2 -NWs with solid structure and approximate diameter of 10nm formed an interconnected open pore along CP surface through SEM and TEM images. After stable operation 3 cycles, the maximum power density output of MFC used TiO2 -NWs/CP as anode was 392mWm−2, this value increased by 49.5% compared to that of the raw CP (198mWm−2). Cyclic voltammetry (CV) analysis indicated TiO2 -NWs could mediate direct extracellular electron transfer (EET) between the bacterium and the electrode, a pair of redox peaks with midpoint potential Em=−0.27V showed biofilm formation on the surface of TiO2 -NWs/CP electrode. Smaller semicircle at the high frequency region represented the lower charge-transfer resistance from electrochemical impedance spectroscopy (EIS) measurement, which was advantageous for promoting electron transfer corresponding to the higher current density output in characterization of CV. In this work, TiO2 -NWs/CP electrode improved the MFC performance owing to the high specific surface area, good biocompatibility, and electrochemical activities. Thus, the results suggested that it would provide more choices for further developing of MFC.

Thermodynamics of the Electron Acceptors in Heliobacterium modesticaldum: An Exemplar of an Early Homodimeric Type I Photosynthetic Reaction Center.

The homodimeric type I reaction center in heliobacteria is arguably the simplest known pigment-protein complex capable of conducting (bacterio)chlorophyll-based conversion of light into chemical energy. Despite its structural simplicity, the thermodynamics of the electron transfer cofactors on the acceptor side have not been fully investigated. In this work, we measured the midpoint potential of the terminal [4Fe-4S](2+/1+) cluster (FX) in reaction centers from Heliobacterium modesticaldum. The FX cluster was titrated chemically and monitored by (i) the decrease in the level of stable P800 photobleaching by optical spectroscopy, (ii) the loss of the light-induced g ≈ 2 radical from P800(+•) following a single-turnover flash, (iii) the increase in the low-field resonance at 140 mT attributed to the S = (3)/2 ground spin state of FX(-), and (iv) the loss of the spin-correlated P800(+) FX(-) radical pair following a single-turnover flash. These four techniques led to similar estimations of the midpoint potential for FX of -502 ± 3 mV (n = 0.99), -496 ± 2 mV (n = 0.99), -517 ± 10 mV (n = 0.65), and -501 ± 4 mV (n = 0.84), respectively, with a consensus value of -504 ± 10 mV (converging to n = 1). Under conditions in which FX is reduced, the long-lived (∼15 ms) P800(+) FX(-) state is replaced by a rapidly recombining (∼15 ns) P800(+)A0(-) state, as shown by ultrafast optical experiments. There was no evidence of the presence of a P800(+) A1(-) spin-correlated radical pair by electron paramagnetic resonance (EPR) under these conditions. The midpoint potentials of the two [4Fe-4S](2+/1+) clusters in the low-molecular mass ferredoxins were found to be -480 ± 11 mV/-524 ± 13 mV for PshBI, -453 ± 6 mV/-527 ± 6 mV for PshBII, and -452 ± 5 mV/-533 ± 8 mV for HM1_2505 as determined by EPR spectroscopy. FX is therefore suitably poised to reduce one [4Fe-4S](2+/1+) cluster in these mobile electron carriers. Using the measured midpoint potential of FX and a quasi-equilibrium model of charge recombination, the midpoint potential of A0 was estimated to be -854 mV at room temperature. The midpoint potentials of A0 and FX are therefore 150-200 mV less reducing than their respective counterparts in Photosystem I of cyanobacteria and plants. This places the redox potential of the FX cluster in heliobacteria approximately equipotential to the highest-potential iron-sulfur cluster (FA) in Photosystem I, consistent with its assignment as the terminal electron acceptor.

Using Electrochemical Techniques to Elucidate Electron-Transport Mechanisms in Microbial Anode Respiration

Anode-respiring bacteria (ARB) catalyze the complete oxidation of organic compounds (e.g. acetate, glucose) into electrical current and carbon dioxide. ARB naturally produce a biofilm at the electrode surface of up to 100 micrometers, where even cells on the outer part of the biofilm are participating in current production. The specific pathway by which ARB transport electrons intra- and extracellularly is still largely unknown. This is largely due to the fact that many ARB have redundant respiratory mechanisms, leading to a large set of possible proteins involved in these mechanisms. Our research utilizes a variety of electrochemical techniques in order to characterize electron transport responses from various ARB. Our goal is to provide insights into the possible mechanisms of electron transfer from the electrochemical responses of ARB biofilms. Through these experiments, we have observed a complex response to anode potential that allows ARB, such as G. sulfurreducens, to optimize their efficiency in electron transport. In G. sulfurreducens, two pathways were identified for anode respiration with midpoint potentials of – 0.155 ±0.005 and –0.095 ± 0.003 V versus SHE. G. sulfurreducens can shift between these two pathways depending on the anode potential in order to optimize its energy recovery from anode respiration. Pathway shifts were observed within 5-20 minutes of a shift in anode potential, suggesting that this microorganism has a potential-sensing mechanism. The fast shifts also confirm that the slow-scan cyclic voltammograms (~1 mV/s or slower) commonly performed in our field can be the result of transient responses by ARB, who can shift pathways within the experimental time. Multiple redox pathways have also been observed in thermophile T. ferriacetica and alkaliphile Geoalkalibacter ferrihydriticus, suggesting that most ARB can have multiple anode-respiration pathways. While the topic of electron transport is the focus of most ARB research, ionic transport is an important factor in determining rate-limiting and potential loss processes. ARB require near-neutral pH in the medium to grow, differing from chemical fuel cells commonly employed, which run under acidic or alkaline conditions. This pH requirement results in a major transport limitation, as H+ ions (now in mM range) should be transported from anode to cathode to achieve electron neutrality. In an MXC anode, H+ ions accumulate in the ARB biofilm, creating an acidification that limits current generation. This behavior has been observed with several ARB, including T. ferriacetica and Glk. ferrihydriticus. Through these experiments, we determined that the rate-limiting protein that is responsible for the potential response of ARB is a proton-coupled reaction. Experiments at different pH values result in shifts in the midpoint potential response of ARB, suggesting a proton coupled redox reaction. The specific stoichiometry of the reaction is difficult to elucidate since the H+ accumulation in the biofilm causes a pH gradient. Nonetheless, our studies provide insights into the characteristics of the rate-limiting proteins in anode respiration which are yet to be identified.

Understanding Electron Transport in a Durable Mixed Community Microbial Biocathode Enriched from Seawater

Biocathode MCL (Marinobacter, Chromatiaceae, Labrenzia) is a cathode associated microbial consortium enriched from seawater.(1-4) It is extremely durable, having been maintained in the laboratory for at least 6 years with minimal care, and acquires energy by coupling electron uptake from graphite or gold electrodes with reduction of O2, directing a portion of the acquired electrons and energy to reduce CO2 for cell growth. Here we report electrochemical gating measurements performed on Biocathode MCL grown on interdigitated microelectrode arrays (IDAs) to characterize its extracellular electron transport (EET) properties in the same manner recently utilized for Geobacter sulfurreducens.(5-9) These measurements revealed a peak-shaped dependency of electrical conductivity on gate potential, consistent with redox conduction (i.e., electron hopping). The maximum biofilm conductivity at 30°C is 60 µS/cm, more than 10-fold greater than that of G. sulfurreducens biofilms.(5) In addition, conductivity of Biocathode MCL decreases with decreasing temperature in an Arrhenius manner also consistent with redox conduction. The apparent reorganization energy (λ = 0.58 ± 0.015 eV) is similar to that of G. sulfurreducens, suggesting hemes act as the EET cofactors. Voltammetric peaks attributable to EET cofactors are not observed under non-turnover condition (oxygen removed) however, indicating relatively low cofactor abundance. The apparent cofactor formal potential, indicated by the conductivity vs. gate potential dependency, is 0.14 V positive of the O2 reduction catalytic midpoint potential, indicating that the consortium utilizes electrons for oxygen reduction at potentials for which the majority of EET cofactors are reduced and the biofilm possesses only a fraction of its maximum conductivity. Confocal microscopy and fluorescent in situ hybridization indicate that Biocathode MCL biofilms are spatially organized such that the putative electroautotrophic organism is localized at the electrode surface. 16S rRNA gene sequencing analysis indicates that the community composition is stable on different electrode materials (gold or graphite electrodes). Finally, an unidentified iron-sulfur protein was detected by confocal resonance Raman microscopy (CRRM). We study biocathode MCL with the long-term goal of gaining mechanistic insights for engineering rugged and efficient electrode catalysts for electrosynthesis of liquid fuel precursors from CO2 accumulating in seawater. The results reported her expand upon our fundamental understanding of its EET properties. 1. Wang Z, et al. (2015) A Previously Uncharacterized, Nonphotosynthetic Member of the Chromatiaceae Is the Primary CO2-Fixing Constituent in a Self-Regenerating Biocathode. Applied and Environmental Microbiology 81(2):699-712. 2. Leary DH, et al. (2015) Metaproteomic evidence of changes in protein expression following a change in electrode potential in a robust biocathode microbiome. Proteomics 15(20):3486-3496. 3. Strycharz-Glaven SM, et al. (2013) Electrochemical Investigation of a Microbial Solar Cell Reveals a Nonphotosynthetic Biocathode Catalyst. Applied and Environmental Microbiology 79(13):3933-3942. 4. Malik S, et al. (2009) A self-assembling self-repairing microbial photoelectrochemical solar cell. Energy & Environmental Science 2(3):292-298. 5. Yates MD, et al. (2015) Thermally activated long range electron transport in living biofilms. Physical Chemistry Chemical Physics 17(48):32564-32570. 6. Boyd DA, et al. (2015) Measuring Electron Transport Rates through Electrochemically Active Biofilms Electrochemically-Active Biofilms in Bioelectrochemical Systems: From Laboratory Practice to Date Interpretation, ed Beyenal H (John Wiley and Sons, Inc., New York). 7. Strycharz-Glaven SM, et al. (2014) Electron Transport through Early Exponential-Phase Anode-Grown Geobacter sulfurreducens Biofilms. ChemElectroChem 1(11):1957-1965. 8. Snider RM, Strycharz-Glaven SM, Tsoi SD, Erickson JS, & Tender LM (2012) Long-range electron transport in Geobacter sulfurreducens biofilms is redox gradient-driven. Proceedings of the National Academy of Sciences of the United States of America 109(38):15467-15472. 9. Strycharz-Glaven SM, Snider RM, Guiseppi-Elie A, & Tender LM (2011) On the electrical conductivity of microbial nanowires and biofilms. Energy & Environmental Science 4:4366-4379.

Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes.

Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H–ThnA4–ThnA3–ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H–ThnA4–ThnA3–ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction.

Modeling electron transfer in photosystem I

Nanosecond to millisecond time-resolved absorption spectroscopy has been used to study electron transfer processes in photosystem I particles from Synechocystis sp. PCC 6803 with eight different quinones incorporated into the A1 binding site, at both 298 and 77K. A detailed kinetic model was constructed and solved within the context of Marcus electron transfer theory, and it was found that all of the data could be well described only if the in situ midpoint potentials of the quinones fell in a tightly defined range. For photosystem I with phylloquinone incorporated into the A1 binding site all of the time-resolved optical data is best modeled when the in situ midpoint potential of phylloquinone on the A/B branch is −635/−690mV, respectively. With the midpoint potential of the FX iron sulfur cluster set at −680mV, this indicates that forward electron transfer from A1− to FX is slightly endergonic/exergonic on the A/B branch, respectively. Additionally, for forward electron transfer from A1− to FX, on both the A and B branches the reorganization energy is close to 0.7eV. Reorganization energies of 0.4 or 1.0eV are not possible.For the eight different quinones incorporated, the same kinetic model was used, allowing us to establish in situ redox potentials for all of the incorporated quinones on both branches. A linear correlation was found between the in situ and in vitro midpoint potentials of the quinones on both branches.

Spectroscopic and Electrochemical Characterization of the Iron–Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase

TsrM, an annotated radical S-adenosylmethionine (SAM) enzyme, catalyzes the methylation of carbon 2 of the indole ring of L-tryptophan. Its reaction is the first step in the biosynthesis of the unique quinaldic acid moiety of thiostrepton A, a thiopeptide antibiotic. The appended methyl group derives from SAM; however, the enzyme also requires cobalamin and iron-sulfur cluster cofactors for turnover. In this work we report the overproduction and purification of TsrM and the characterization of its metallocofactors by UV-visible, electron paramagnetic resonance, hyperfine sublevel correlation (HYSCORE), and Mössbauer spectroscopies as well as protein-film electrochemistry (PFE). The enzyme contains 1 equiv of its cobalamin cofactor in its as-isolated state and can be reconstituted with iron and sulfide to contain one [4Fe-4S] cluster with a site-differentiated Fe(2+)/Fe(3+) pair. Our spectroscopic studies suggest that TsrM binds cobalamin in an uncharacteristic five-coordinate base-off/His-off conformation, whereby the dimethylbenzimidazole group is replaced by a non-nitrogenous ligand, which is likely a water molecule. Electrochemical analysis of the protein by PFE indicates a one-electron redox feature with a midpoint potential of -550 mV, which is assigned to a [4Fe-4S](2+)/[4Fe-4S](+) redox couple. Analysis of TsrM by Mössbauer and HYSCORE spectroscopies suggests that SAM does not bind to the unique iron site of the cluster in the same manner as in other radical SAM (RS) enzymes, yet its binding still perturbs the electronic configuration of both the Fe/S cluster and the cob(II)alamin cofactors. These biophysical studies suggest that TsrM is an atypical RS enzyme, consistent with its reported inability to catalyze formation of a 5'-deoxyadenosyl 5'-radical.

An Explicit Physics-Based I-V Model for Surrounding-Gate Polysilicon Transistors

An explicit current–voltage model for cylindrical surrounding-gate polysilicon (poly-Si) transistors with a doped poly-Si channel is presented. It is based on an intermediate parameter which is derived analytically from the 1-D Poisson’s equation and satisfies the corresponding boundary conditions. The surface and midpoint potentials are described as functions of this intermediate parameter. Furthermore, the drain currents in the different operational regions are analytically integrated. Our model results show good agreement with numerical and experimental data. The use of the explicit surface potential solution results in an easy implementation into circuit simulators.

Tuning of Hemes b Equilibrium Redox Potential Is Not Required for Cross-Membrane Electron Transfer

In biological energy conversion, cross-membrane electron transfer often involves an assembly of two hemes b. The hemes display a large difference in redox midpoint potentials (ΔEm_b), which in several proteins is assumed to facilitate cross-membrane electron transfer and overcome a barrier of membrane potential. Here we challenge this assumption reporting on heme b ligand mutants of cytochrome bc1 in which, for the first time in transmembrane cytochrome, one natural histidine has been replaced by lysine without loss of the native low spin type of heme iron. With these mutants we show that ΔEm_b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔEm_b can be markedly decreased to the point that two hemes are almost isopotential, yet the enzyme retains catalytically competent electron transfer between quinone binding sites and remains functional in vivo. This reveals that cytochrome bc1 can accommodate large changes in ΔEm_b without hampering catalysis, as long as these changes do not impose overly endergonic steps on downhill electron transfer from substrate to product. We propose that hemes b in this cytochrome and in other membranous cytochromes b act as electronic connectors for the catalytic sites with no fine tuning in ΔEm_b required for efficient cross-membrane electron transfer. We link this concept with a natural flexibility in occurrence of several thermodynamic configurations of the direction of electron flow and the direction of the gradient of potential in relation to the vector of the electric membrane potential.