Flavin Redox Bifurcation as a Mechanism for Controlling the Direction of Electron Flow during Extracellular Electron Transfer

The iron‐reducing bacterium Shewanella oneidensis MR‐1 has a dual directional electronic conduit involving 40 heme redox centers in flavin‐binding outer‐membrane c‐type cytochromes (OM c‐Cyts). While the mechanism for electron export from the OM c‐Cyts to an anode is well understood, how the redox centers in OM c‐Cyts take electrons from a cathode has not been elucidated at the molecular level. Electrochemical analysis of live cells during switching from anodic to cathodic conditions showed that altering the direction of electron flow does not require gene expression or protein synthesis, but simply redox potential shift about 300 mV for a flavin cofactor interacting with the OM c‐Cyts. That is, the redox bifurcation of the riboflavin cofactor in OM c‐Cyts switches the direction of electron conduction in the biological conduit at the cell–electrode interface to drive bacterial metabolism as either anode or cathode catalysts.     

Supercapacitors Based on c‐Type Cytochromes Using Conductive Nanostructured Networks of Living Bacteria

Supercapacitors have attracted interest in energy storage because they have the potential to complement or replace batteries. Here, we report that c‐type cytochromes, naturally immersed in a living, electrically conductive microbial biofilm, greatly enhance the device capacitance by over two orders of magnitude. We employ genetic engineering, protein unfolding and Nernstian modeling for in vivo demonstration of charge storage capacity of c‐type cytochromes and perform electrochemical impedance spectroscopy, cyclic voltammetry and charge–discharge cycling to confirm the pseudocapacitive, redox nature of biofilm capacitance. The biofilms also show low self‐discharge and good charge/discharge reversibility. The superior electrochemical performance of the biofilm is related to its high abundance of cytochromes, providing large electron storage capacity, its nanostructured network with metallic‐like conductivity, and its porous architecture with hydrous nature, offering prospects for future low cost and environmentally sustainable energy storage devices.     

Distinct Electron Conductance Regimes in Bacterial Decaheme Cytochromes

Shewanella oneidensis MR‐1 gains energy by extracellular electron transfer to solid surfaces. They employ c‐type cytochromes in two Mtr transmembrane complexes, forming a multiheme wire for electron transport across the cellular outer membrane. We investigated electron‐ and hole‐transfer mechanisms in the external terminal of the two complexes, MtrC and MtrF. Comparison of computed redox potentials with previous voltammetry experiments in distinct environments (isolated and electrode‐bound conditions of PFV or in vivo) suggests that these systems function in different regimes depending on the environment. Analysis of redox potential shifts in different regimes indicates strong coupling between the hemes via an interplay between direct Coulomb and indirect interactions through local structural reorganization. The latter results in the screening of Coulomb interactions and explains poor correlation of the strength of the heme‐to‐heme interactions with the distance between the hemes.     

Superior Electron‐Transport Ability of π‐Conjugated Redox Molecular Wires Prepared by the Stepwise Coordination Method on a Surface

Electronic conductivity of molecular wires is a critical fundamental issue in molecular electronics. π‐Conjugated redox molecular wires with the superior long‐range electron‐transport ability could be constructed on a gold surface through the stepwise ligand–metal coordination method. The β^d value, indicating the degree of decrease in the electron‐transfer rate constant with distance along the molecular wire between the electrode and the redox active species at the terminal of the wire, were 0.008–0.07 Å^?1 and 0.002–0.004 Å^?1 for molecular wires of bis(terpyridine)iron and bis(terpyridine)cobalt complex oligomers, respectively. The influences on β^d by the chemical structure of molecular wires and the terminal redox units, temperature, electric field, and electrolyte concentration were clarified. The results indicate that facile sequential electron hopping between neighboring metal–complex units within the wire is responsible for the high electron‐transport ability.     

Functionalization of gold electrode surface with heterobifunctional poly(ethylene oxide)s having both mercapto and aldehyde groups

We synthesized heterobifunctional poly(ethylene oxide) (PEO) (α‐formyl‐ω‐mercapto‐PEO; CHO‐PEO₄₀₀‐SH, average molecular weight of PEO part being 400), which had both an aldehyde group as a binding site with amino group of protein and a mercapto group for gold electrode surface. The CHO‐PEO₄₀₀‐SH was adsorbed on a gold electrode surface and cytochrome c (cyt.c) was fixed on this modified electrode. The redox response of covalently immobilized cyt.c was observed on the cyclic voltammetry measurement, showing that CHO‐PEO₄₀₀‐SH can be used as a linker to fix cyt.c on an electrode. Another type of heterobifunctional PEO (α‐formyl‐ω‐(2‐pyridyldithio)‐PEO; CHO‐PEO₃₀₀‐SS‐Py), which had an aldehyde group and a 2‐pyridinethiol (2‐Py) through disulfide bond, was synthesized to form co‐adsorbed monolayer of PEO chain and 2‐Py on an electrode surface. It was expected, due to the spacer with shorter PEO chain and lower surface density, that better redox response of the fixed cyt.c was obtained. However, the redox response of fixed cyt.c was not detected on the CHO‐PEO₃₀₀‐SS‐Py modified gold electrode. Instead, this heterobifunctional PEO was found to function as a good promoter for cyt.c dissolved in phosphate buffer solution. Copyright © 2003 John Wiley & Sons, Ltd.     

Wettability‐Regulated Extracellular Electron Transfer from the Living Organism of Shewanella loihica PV‐4

C‐type cytochromes located on the outer membrane (OMCs) of genus Shewanella act as the main redox‐active species to mediate extracellular electron transfer (EET) from the inside of the outer membrane to the external environment: the central challenge that must be met for successful EET. The redox states of OMCs play a crucial role in dictating the rate and extent of EET. Here, we report that the surface wettability of the electrodes strongly influences the EET activity of living organisms of Shewanella loihica PV‐4 at a fixed external potential: the EET activity on a hydrophilic electrode is more than five times higher than that on a hydrophobic one. We propose that the redox state of OMCs varies significantly at electrodes with different wettability, resulting in different EET activities.     

Creating a Low‐Potential Redox Polymer for Efficient Electroenzymatic CO2 Reduction

Increasing greenhouse gas emissions have resulted in greater motivation to find novel carbon dioxide (CO₂) reduction technologies, where the reduction of CO₂ to valuable chemical commodities is desirable. Molybdenum‐dependent formate dehydrogenase (Mo‐FDH) from Escherichia coli is a metalloenzyme that is able to interconvert formate and CO₂. We describe a low‐potential redox polymer, synthesized by a facile method, that contains cobaltocene (grafted to poly(allylamine), Cc‐PAA) to simultaneously mediate electrons to Mo‐FDH and immobilize Mo‐FDH at the surface of a carbon electrode. The resulting bioelectrode reduces CO₂ to formate with a high Faradaic efficiency of 99±5 % at a mild applied potential of ?0.66 V vs. SHE.     

Intrachain versus Interchain Electron Transport in Poly(fluorene‐alt‐benzothiadiazole): A Quantum‐Chemical Insight

Poly(9,9‐di‐n‐octylfluorene‐alt‐benzothiadiazole) [F8BT], displays very different charge‐transport properties for holes versus electrons when comparing annealed and pristine thin films and transport parallel (intrachain) and perpendicular (interchain) to the polymer axes. The present theoretical contribution focuses on the electron‐transport properties of F8BT chains and compares the efficiency of intrachain versus interchain transport in the hopping regime. The theoretical results rationalize significantly lowered electron mobility in annealed F8BT thin films and the smaller mobility anisotropy (μ_∥/μ_⊥) measured for electrons in aligned films (i.e. 5–7 compared to 10–15 for holes).     

Thermodynamics of electron flow in the bacterial deca-heme cytochrome MtrF

Electron-transporting multi-heme cytochromes are essential to the metabolism of microbes that inhabit soils and carry out important biogeochemical processes. Recently the first crystal structure of a prototype bacterial deca-heme cytochrome (MtrF) has been resolved and its electrochemistry characterized. However, the molecular details of electron transport along heme chains in the cytochrome are difficult to access via experiment due to the nearly identical chemical nature of the heme cofactors. Here we employ large-scale molecular dynamics simulations to compute the redox potentials of the 10 hemes of MtrF in aqueous solution. We find that as a whole they fall within a range of ~0.3 V, in agreement with experiment. Individual redox potentials give rise to a free energy profile for electron transport that is approximately symmetric with respect to the center of the protein. Our calculations indicate that there is no significant potential bias along the orthogonal octa- and tetra-heme chains, suggesting that under aqueous conditions MtrF is a nearly reversible two-dimensional conductor.     

Bioelectrocatalysis by Microperoxidase‐11 in a Multilayer Architecture of Chitosan Embedded Gold Nanoparticles

We report on the redox behaviour of the microperoxidase‐11 (MP‐11) which has been electrostatically immobilized in a matrix of chitosan‐embedded gold nanoparticles on the surface of a glassy carbon electrode. MP‐11 contains a covalently bound heme c as the redox active group that exchanges electrons with the electrode via the gold nanoparticles. Electroactive surface concentration of MP‐11 at high scan rate is between 350±50 pmol cm^?2, which reflects a multilayer process. The formal potential (E°′) of MP‐11 in the gold nanoparticles‐chitosan film was estimated to be ?(267.7±2.9) mV at pH 7.0. The heterogeneous electron transfer rate constant (k_s) starts at 1.21 s^?1 and levels off at 6.45 s^?1 in the scan rate range from 0.1 to 2.0 V s^?1. Oxidation and reduction of MP‐11 by hydrogen peroxide and superoxide, respectively have been coupled to the direct electron transfer of MP‐11.     

Kinetics and Mechanism of Mineral Respiration: How Iron Hemes Synchronize Electron Transfer Rates

Anaerobic microorganisms of the Geobacter genus are effective electron sources for the synthesis of nanoparticles, for bioremediation of polluted water, and for the production of electricity in fuel cells. In multistep reactions, electrons are transferred via iron/heme cofactors of c‐type cytochromes from the inner cell membrane to extracellular metal ions, which are bound to outer membrane cytochromes. We measured electron production and electron flux rates to 5×10⁵ e s^?1 per G. sulfurreducens. Remarkably, these rates are independent of the oxidants, and follow zero order kinetics. It turned out that the microorganisms regulate electron flux rates by increasing their Fe²⁺/Fe³⁺ ratios in the multiheme cytochromes whenever the activity of the extracellular metal oxidants is diminished. By this mechanism the respiration remains constant even when oxidizing conditions are changing. This homeostasis is a vital condition for living systems, and makes G. sulfurreducens a versatile electron source.     

Redox and Conformational Equilibria and Dynamics of Cytochrome c at High Electric Fields

Cytochrome c (Cyt‐c) adsorbed in the electrical double layer of the Ag electrode/electrolyte interface has been studied by stationary and time‐resolved surface‐enhanced resonance Raman spectroscopy to analyse the effect of strong electric fields on structure and reaction equilibria and dynamics of the protein. In the potential range between +0.1 and ?0.55 V (versus saturated calomel electrode), the adsorbed Cyt‐c forms a potential‐dependent reversible equilibrium between the native state B1 and a conformational state B2. The redox potentials of the bis‐histidine‐coordinated six‐coordinated low‐spin and five‐coordinated high‐spin substates of B2 were determined to be ?0.425 and ?0.385 V, respectively, whereas the additional six‐coordinated aquo‐histidine‐coordinated high‐spin substate was found to be redox‐inactive. The redox potential for the conformational state B1 was found to be the same as in solution in agreement with the structural identity of the adsorbed B1 and the native Cyt‐c. For all three redox‐active species, the formal heterogeneous electron transfer rate constants are small and of the same order of magnitude (3–13 s^?1), which implies that the rate‐limiting step is largely independent of the redox‐site structure. These findings, as well as the slow and potential‐dependent transitions between the various conformational (sub‐)states, can be rationalized in terms of an electric field‐induced increase of the activation energy for proton‐transfer steps linked to protein structural reorganisation. Further increasing the electric field strength by shifting the electrode potential above +0.1 V leads to irreversible structural changes that are attributed to an unfolding of the polypeptide chain.     

UV-ozone dry-cleaning process for indium oxide electrodes for protein electrochemistry

The present study reports, for the first time, on electrochemical responses of cytochrome c at a UV-ozone treated indium oxide electrode. Results from surface tension measurements indicate that UV-ozone treatment is an efficient cleaning procedure to remove organic species contamination on surfaces. Well-defined redox responses for cytochrome c were observed at a UV-ozone treated fully hydrophilic indium oxide electrode. Electrochemical parameters, including the diffusion coefficient, the heterogeneous electron transfer rate constant and the redox potential, were in good agreement with those previously reported. However, decrease in peak current for cytochrome c and [Fe(CN)₆]⁴⁻ were observed at a UV-ozone treated electrode. From XPS results, this behavior would be understood to indicate a decrease in homogeneous active electrode surface area by a decrease in conductivity of the indium oxide surface by UV-ozone treatment. Simple and effective UV-ozone treatment methods are useful for surface contamination sensitive electrochemistry.     

Comparative Quantum-Chemical Analysis of the Electronic Structures of the Valence Regions of Active Sites in Cytochromes f and c.

A comparative analysis of densities of states has been carried out for the valence regions of the hemes and clusters of cytochromes f and c using the ZINDO1 semiempirical quantum-chemical method. The molecular orbitals of these structures are formed from the p atomic orbitals of nitrogen and carbon of the porphyrin ring, making equal contributions. For systems with negative charges, more than half of all added electrons are distributed over the porphyrin parts of the molecules. The molecular orbital energies of the valence regions of the corresponding clusters and hemes are nearly the same. In iron porphyrin, as well as in heme f and cytochrome cluster f, the lowest unoccupied molecular orbital is doubly degenerate. In heme c and cytochrome cluster c, the degeneracy is lifted because of the asymmetry of the nearest aminoacid environment and substituents in the porphyrin ring.     

Direct Electrochemistry of Cytochrome c Based on Poly(diallyldimethylammonium Chloride)‐ Graphene Nanosheets/Gold Nanoparticles Hybrid Nanocomposites and Its Biosensing

A novel biosensor was developed by entrapping cytochrome c (Cyt c) in thin films of the room temperature ionic liquid (RTIL) containing nanocomposites of poly(diallyldimethylammonium chloride)‐graphene nanosheets‐gold nanoparticles (PDDA‐Gp‐AuNPs) at a 11‐mercaptoundecanoic acid‐6‐mercapto‐1‐hexanol modified gold electrode. The synthesized PDDA‐Gp‐AuNPs hybrid nanocomposites were characterized by UV‐vis spectroscopy, Raman spectroscopy, scanning electron microscopy and atomic force microscopy. The PDDA‐Gp‐AuNPs nanocomposites could increase the effective surface of the electrode, enhance the fixed amount of Cyt c on the electrode surface, promote the electron transfer and facilitate the catalytic activity of Cyt c. The RTIL could provide a biocompatible microenvironment to keep Cyt c biological activities, act as an effective mediator to immobilize a large number of Cyt c on the electrode and have good conductivity to improve electron transfer. Therefore, the resultant electrode exhibited good electrochemical performance and electrocatalytic activity. It could be used for electrochemical detection of H₂O₂ with rapid response, high sensitivity, wide linear range and low detection limit, as well as good stability, repeatability and selectivity. The sensor might be promising for practical application.     

Long Distance Electron Transfer Across >100 nm Thick Au Nanoparticle/Polyion Films to a Surface Redox Protein

Glutathione‐decorated 5 nm gold nanoparticles (AuNPs) and oppositely charged poly(allylamine hydrochloride) (PAH) were assembled into {PAH/AuNP}_n films fabricated layer‐by‐layer (LbL) on pyrolytic graphite (PG) electrodes. These AuNP/polyion films utilized the AuNPs as electron hopping relays to achieve direct electron transfer between underlying electrodes and redox proteins on the outer film surface across unprecedented distances >100 nm for the first time. As film thickness increased, voltammetric peak currents for surface myoglobin (Mb) on these films decreased but the electron transfer rate was relatively constant, consistent with a AuNP‐mediated electron hopping mechanism.     

Physicochemical Characterization of Acidiphilium sp. Biofilms

The biofilm formation of a strain of the extremophile bacterium Acidiphilium sp., capable of donating electrons directly to electrodes, was studied by different surface characterization techniques. We develop a method that allows the simultaneous study of bacterial biofilms by means of fluorescence microscopy and atomic force microscopy (AFM), in which transparent graphitic flakes deposited on a glass substrate are used as a support for the biofilm. The majority of the cells present on the surface were viable, and the growth of the biofilms over time showed a critical increase of the extracellular polymeric substances (EPS) as well as the formation of nanosized particles inside the biofilm. Also, the presence of Fe in Acidiphilium biofilms was determined by X‐ray photoelectron spectroscopy (XPS), whereas surface‐enhanced infrared absorption spectroscopy indicated the presence of redox‐active proteins.     

Electorchemical Studies of Cytochrome c on Electodes Modified by Single—Wall Carbon Naotubes

Single-wall carbon nanotubes(SWNTs) modified gold electrodes were prepared by using two different methods.The electrochemical behavior of cytochrome c on the modified gold electrodes was investigated.The first kind of SWNT-modified electrode (noted as SWNT/Au electrode)was prepared by the adsorption of carboxylterminated SWNTs from DMF dispersion on the gold electrode.The oxidatively processed SWNT tips were covalently modified by coupling with amines (AET) to form amide linkage.Via Au-S chemical bonding,the self-assembled monolayer of thiol-unctionalized nanotubes on gold surface was fabricated so as to prepare the others SWNT-modified electrode (noted as SWNT/AET/Au electrode).It was shown from cyclic voltammetry cxperiments that cytochrome c exhibited direct electrochemical responses on the both electrodes, but only the current of controlled diffusion existed on the SWNT/Au electrode while both the currents of controlled diffusion and adsorption of cytochrome c occurred on the SWNT/AET/Au electrode.Photoelastic Modulation Infared Reflection Absorpthion Spectroscopy (PEM-IRRAS) and Quartz Crystal Microbalance (QCM) were employed to verify the adsorption of SWNTs on the gold electrodes.The results proved that SWNTs could enhance the direct electron transfer proecss between the electrodes and redox proteins.     

Synthesis of Molecular Wires of Linear and Branched Bis(terpyridine)‐Complex Oligomers and Electrochemical Observation of Through‐Bond Redox Conduction

Films of linear and branched oligomer wires of Fe(tpy)₂ (tpy=2,2′:6′,2′′‐terpyridine) were constructed on a gold‐electrode surface by the interfacial stepwise coordination method, in which a surface‐anchoring ligand, (tpy? C₆H₄N?NC₆H₄? S)₂ ( 1 ), two bridging ligands, 1,4‐(tpy)₂C₆H₄ ( 3 ) and 1,3,5‐(C?C? tpy)₃C₆H₃ ( 4 ), and metal ions were used. The quantitative complexation of the ligands and Fe^II ions was monitored by electrochemical measurements in up to eight complexation cycles for linear oligomers of 3 and in up to four cycles for branched oligomers of 4 . STM observation of branched oligomers at low surface coverage showed an even distribution of nanodots of uniform size and shape, which suggests the quantitative formation of dendritic structures. The electron‐transport mechanism and kinetics for the redox reaction of the films of linear and branched oligomer wires were analyzed by potential‐step chronoamperometry (PSCA). The unique current‐versus‐time behavior observed under all conditions indicates that electron conduction occurs not by diffusional motion but by successive electron hopping between neighboring redox sites within a molecular wire. Redox conduction in a single molecular wire in a redox‐polymer film has not been reported previously. The analysis provided the rate constant for electron transfer between the electrode and the nearest redox‐complex moiety, k₁ (s^?1), as well as that for intrawire electron transfer between neighboring redox‐complex moieties, k₂ (cm² mol^?1 s^?1). The strong effect of the electrolyte concentration on both k₁ and k₂ indicates that the counterion motion limits the electron‐hopping rate at lower electrolyte concentrations. Analysis of the dependence of k₁ and k₂ on the potential gave intrinsic kinetic parameters without overpotential effects: k₁⁰=110 s^?1, k₂⁰=2.6×10¹² cm² mol^?1 s^?1 for [n Fe 3 ], and k₁⁰=100 s^?1, k₂⁰=4.1×10¹¹ cm² mol^?1 s^?1 for [n Fe 4 ] (n=number of complexation cycles).     

A Four‐Electron Sulfur Electrode Hosting a Cu2+/Cu+ Redox Charge Carrier

The elemental sulfur electrode with Cu²⁺ as the charge carrier gives a four‐electron sulfur electrode reaction through the sequential conversion of S?CuS?Cu₂S. The Cu‐S redox‐ion electrode delivers a high specific capacity of 3044 mAh g^?1 based on the sulfur mass or 609 mAh g^?1 based on the mass of Cu₂S, the completely discharged product, and displays an unprecedently high potential of sulfur/metal sulfide reduction at 0.5 V vs. SHE. The Cu‐S electrode also exhibits an extremely low extent of polarization of 0.05 V and an outstanding cycle number of 1200 cycles retaining 72 % of the initial capacity at 12.5 A g^?1. The remarkable utility of this Cu‐S cathode is further demonstrated in a hybrid cell that employs an Zn metal anode and an anion‐exchange membrane as the separator, which yields an average cell discharge voltage of 1.15 V, the half‐cell specific energy of 547 Wh kg^?1 based on the mass of the Cu₂S/carbon composite cathode, and stable cycling over 110 cycles.