Structure-activity relationships of hierarchical three-dimensional electrodes with photosystem II for semi-artifcial photosynthesis

Semi-artificial photosynthesis integrates photosynthetic enzymes with artificial electronics, which is an emerging approach to reroute the natural photoelectrogenetic pathways for sustainable fuel and chemical synthesis. However, the reduced catalytic activity of enzymes in bioelectrodes limits the overall performance and further applications in fuel production. Here, we show new insights into factors that govern the photoelectrogenesis in a model system consisting of photosystem II and three-dimensional indium tin oxide and graphene electrodes. Fluorescence microscopy and in situ surface-sensitive infrared spectroscopy are employed to probe the enzyme distribution and penetration within electrode scaffolds of different structures, which is further correlated with protein film-photoelectrochemistry to establish relationships between the electrode structure and enzyme activity. We find that the hierarchical 1 structure of electrodes mainly affects the protein integration, but not the enzyme activity. Photoactivity is more limited by light intensity and electronic communication at the biointerface. This study provides guidelines for maximizing the performance of semi-artificial photosynthesis and also presents a set of methodologies to probe the photoactive biofilms in three-dimensional electrodes.CSC-Cambridge PhD Scholarship, EPSRC PhD studentship, Newton-Mosharafa Research Fellowship, ERC Consolidator Grant 'MatEnSAP

Advancing Techniques for Investigating the Enzyme-Electrode Interface.

Enzymes are the essential catalytic components of biology and adsorbing redox-active enzymes on electrode surfaces enables the direct probing of their function. Through standard electrochemical measurements, catalytic activity, reversibility and stability, potentials of redox-active cofactors, and interfacial electron transfer rates can be readily measured. Mechanistic investigations on the high electrocatalytic rates and selectivity of enzymes may yield inspiration for the design of synthetic molecular and heterogeneous electrocatalysts. Electrochemical investigations of enzymes also aid in our understanding of their activity within their biological environment and why they evolved in their present structure and function. However, the conventional array of electrochemical techniques (e.g., voltammetry and chronoamperometry) alone offers a limited picture of the enzyme-electrode interface. How many enzymes are loaded onto an electrode? In which orientation(s) are they bound? What fraction is active, and are single or multilayers formed? Does this static picture change over time, applied voltage, or chemical environment? How does charge transfer through various intraprotein cofactors contribute to the overall performance and catalytic bias? What is the distribution of individual enzyme activities within an ensemble of active protein films? These are central questions for the understanding of the enzyme-electrode interface, and a multidisciplinary approach is required to deliver insightful answers. Complementing standard electrochemical experiments with an orthogonal set of techniques has recently allowed to provide a more complete picture of enzyme-electrode systems. Within this framework, we first discuss a brief history of achievements and challenges in enzyme electrochemistry. We subsequently describe how the aforementioned challenges can be overcome by applying advanced electrochemical techniques, quartz-crystal microbalance measurements, and spectroscopic, namely, resonance Raman and infrared, analysis. For example, rotating ring disk electrochemistry permits the simultaneous determination of reaction kinetics and quantification of generated products. In addition, recording changes in frequency and dissipation in a quartz crystal microbalance allows to shed light into enzyme loading, relative orientation, clustering, and denaturation at the electrode surface. Resonance Raman spectroscopy yields information on ligation and redox state of enzyme cofactors, whereas infrared spectroscopy provides insights into active site states and the protein secondary and tertiary structure. The development of these emerging methods for the analysis of the enzyme-electrode interface is the primary focus of this Account. We also take a critical look at the remaining gaps in our understanding and challenges lying ahead toward attaining a complete mechanistic picture of the enzyme-electrode interface.Royal Society Newton International Fellowship, European Research Council (ERC) Consolidator Grant (H2020), Marie Sklodowska-Curie Individual Fellowshi

Microporous polymer network films covalently bound to gold electrodes

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.Covalent attachment of a microporous polymer network (MPN) on a gold surface is presented. A functional bromophenyl-based self-assembled monolayer (SAM) formed on the gold surface acts as co-monomer in the polymerisation of the MPN yielding homogeneous and robust coatings. Covalent binding of the films to the electrode is confirmed by SEIRAS measurements.EC/FP7/278593/EU/Organic Zeolites/ORGZEODFG, EXC 314, Unifying Concepts in CatalysisBMBF, 03X5524, DELKAT - Hydrophobe Nanoreaktor Templatierung - Eine Tool-Box für optimierte Elektrokatalysatore

Disparity of Cytochrome Utilization in Anodic and Cathodic Extracellular Electron Transfer Pathways of Geobacter sulfurreducens Biofilms.

Extracellular electron transfer (EET) in microorganisms is prevalent in nature and has been utilized in functional bioelectrochemical systems. EET of Geobacter sulfurreducens has been extensively studied and has been revealed to be facilitated through c-type cytochromes, which mediate charge between the electrode and G. sulfurreducens in anodic mode. However, the EET pathway of cathodic conversion of fumarate to succinate is still under debate. Here, we apply a variety of analytical methods, including electrochemistry, UV-vis absorption and resonance Raman spectroscopy, quartz crystal microbalance with dissipation, and electron microscopy, to understand the involvement of cytochromes and other possible electron-mediating species in the switching between anodic and cathodic reaction modes. By switching the applied bias for a G. sulfurreducens biofilm coupled to investigating the quantity and function of cytochromes, as well as the emergence of Fe-containing particles on the cell membrane, we provide evidence of a diminished role of cytochromes in cathodic EET. This work sheds light on the mechanisms of G. sulfurreducens biofilm growth and suggests the possible existence of a nonheme, iron-involving EET process in cathodic mode.N.K. was supported by a Royal Society Newton International Fellowship, NF160054. E.R., X.F. and N.H. acknowledge the European Research Council (ERC) Consolidator Grant “MatEnSAP” (682833). S. K. was supported by a Marie Skłodowska-Curie Fellowship (EMES, 744317). K. H. Ly acknowledges the Open Topic Postdoc Programme of the Technische Universität Dresden and the Marie Sklodowska Curie IF, GAN 701192. The TEM was funded through the EPSRC underpinning multi-user equipment call (EP/P030467/1

Disparity of Cytochrome Utilization in Anodic and Cathodic Extracellular Electron Transfer Pathways of Geobacter sulfurreducens Biofilms.

Extracellular electron transfer (EET) in microorganisms is prevalent in nature and has been utilized in functional bioelectrochemical systems. EET of Geobacter sulfurreducens has been extensively studied and has been revealed to be facilitated through c-type cytochromes, which mediate charge between the electrode and G. sulfurreducens in anodic mode. However, the EET pathway of cathodic conversion of fumarate to succinate is still under debate. Here, we apply a variety of analytical methods, including electrochemistry, UV-vis absorption and resonance Raman spectroscopy, quartz crystal microbalance with dissipation, and electron microscopy, to understand the involvement of cytochromes and other possible electron-mediating species in the switching between anodic and cathodic reaction modes. By switching the applied bias for a G. sulfurreducens biofilm coupled to investigating the quantity and function of cytochromes, as well as the emergence of Fe-containing particles on the cell membrane, we provide evidence of a diminished role of cytochromes in cathodic EET. This work sheds light on the mechanisms of G. sulfurreducens biofilm growth and suggests the possible existence of a nonheme, iron-involving EET process in cathodic mode.N.K. was supported by a Royal Society Newton International Fellowship, NF160054. E.R., X.F. and N.H. acknowledge the European Research Council (ERC) Consolidator Grant “MatEnSAP” (682833). S. K. was supported by a Marie Skłodowska-Curie Fellowship (EMES, 744317). K. H. Ly acknowledges the Open Topic Postdoc Programme of the Technische Universität Dresden and the Marie Sklodowska Curie IF, GAN 701192. The TEM was funded through the EPSRC underpinning multi-user equipment call (EP/P030467/1

Interfacing Formate Dehydrogenase with Metal Oxides for the Reversible Electrocatalysis and Solar-Driven Reduction of Carbon Dioxide

The integration of enzymes with synthetic materials allows efficient electrocatalysis and production of solar fuels. Here, we couple formate dehydrogenase (FDH) from Desulfovibrio vulgaris Hildenborough (DvH) to metal oxides for catalytic CO2 reduction and report an in‐depth study of the resulting enzyme–material interface. Protein film voltammetry (PFV) demonstrates the stable binding of FDH on metal‐oxide electrodes and reveals the reversible and selective reduction of CO2 to formate. Quartz crystal microbalance (QCM) and attenuated total reflection infrared (ATR‐IR) spectroscopy confirm a high binding affinity for FDH to the TiO2 surface. Adsorption of FDH on dye‐sensitized TiO2 allows for visible‐light‐driven CO2 reduction to formate in the absence of a soluble redox mediator with a turnover frequency (TOF) of 11±1 s−1. The strong coupling of the enzyme to the semiconductor gives rise to a new benchmark in the selective photoreduction of aqueous CO2 to formate.H2020 European Research Council. Grant Number: MatEnSAP (682833) Royal Society. Grant Number: NF160054 Christian Doppler Forschungsgesellschaft. Grant Number: Sustainable SynGas Chemistry H2020 Fast Track to Innovation. Grant Number: GA 81085

Solar Water Splitting with a Hydrogenase Integrated in Photoelectrochemical Tandem Cells

Hydrogenases (H2ases) are benchmark electrocatalysts for H2 production, both in biology and (photo)catalysis in vitro. We report the tailoring of a p-type Si photocathode for optimal loading and wiring of H2ase through the introduction of a hierarchical inverse opal (IO) TiO2 interlayer. This proton-reducing Si j IO-TiO2 j H2ase photocathode is capable of driving overall water splitting in combination with a photoanode. We demonstrate unassisted (bias-free) water splitting by wiring Si j IO-TiO2 j H2ase to a modified BiVO4 photoanode in a photoelectrochemical (PEC) cell during several hours of irradiation. Connecting the Si j IO-TiO2 j H2ase to a photosystem II (PSII) photoanode provides proof of concept for an engineered Z-scheme that replaces the non-complementary, natural light absorber photosystem I with a complementary abiotic silicon photocathode

Host-Guest Chemistry Meets Electrocatalysis: Cucurbit[6]uril on a Au Surface as a Hybrid System in CO2 Reduction.

The rational control of forming and stabilizing reaction intermediates to guide specific reaction pathways remains to be a major challenge in electrocatalysis. In this work, we report a surface active-site engineering approach for modulating electrocatalytic CO2 reduction using the macrocycle cucurbit[6]uril (CB[6]). A pristine gold surface functionalized with CB[6] nanocavities was studied as a hybrid organic-inorganic model system that utilizes host-guest chemistry to influence the heterogeneous electrocatalytic reaction. The combination of surface-enhanced infrared absorption (SEIRA) spectroscopy and electrocatalytic experiments in conjunction with theoretical calculations supports capture and reduction of CO2 inside the hydrophobic cavity of CB[6] on the gold surface in aqueous KHCO3 at negative potentials. SEIRA spectroscopic experiments show that the decoration of gold with the supramolecular host CB[6] leads to an increased local CO2 concentration close to the metal interface. Electrocatalytic CO2 reduction on a CB[6]-coated gold electrode indicates differences in the specific interactions between CO2 reduction intermediates within and outside the CB[6] molecular cavity, illustrated by a decrease in current density from CO generation, but almost invariant H2 production compared to unfunctionalized gold. The presented methodology and mechanistic insight can guide future design of molecularly engineered catalytic environments through interfacial host-guest chemistry

IR Spektro-Elektrochemie von einer adsorbierten sauerstofftoleranten [NiFe] Hydrogenase

Hydrogenases are metalloenzymes, catalyzing the reversible oxidation of molecular hydrogen into two protons and two electrons. The 'Knallgasbacterium' Ralstonia eutropha H16 (Re) is one of the few examples, harboring several [NiFe] hydrogenases that established mechanisms to enable catalytic conversion of H2 in the presence of O2. In order to exploit the catalytic potential of these oxygen-tolerant hydrogenases, also in view of possible biotechnological applications in biofuel cells, an efficient electronic communication between the enzyme and electrically conducting surface is required. Hence, for optimum performance, an in-depth understanding of the enzyme-surface interactions is needed. This thesis presents an interdisciplinary approach providing detailed insights into the adsorption parameters of heterodimeric membrane-bound [NiFe] hydrogenase (MBH) species of Re on various electrode materials. The first part was dedicated to adsorption studies of oxygen-tolerant MBH on biocompatibly coated Au electrodes by protein film voltammetry (PFV), surface enhanced IR absorption (SEIRA) spectro-electrochemistry, atomic force microscopy (AFM), and ellipsometry in order to elucidate the enzymes catalytic activity, structure, integrity, and surface coverage. Results were obtained for charged and hydrophobic surfaces. Electrocatalytic efficiencies correlated with the mode of protein adsorption on the electrode provided valuable insights in the orientational configuration, which were complemented with molecular dynamics (MD) simulations as well as density functional theory (DFT) calculations, carried out in collaboration. Hence, proposed orientational configurations provided by different accessible binding anchors of the MBH were discussed and experimentally verified by SEIRA spectro-electrochemical investigations. The obtained results suggested that, in particular, an immobilization of heterodimeric MBH on slightly negatively charged surfaces was most beneficial for an efficient interfacial electron transfer (ET). Hereby, the suggested anchoring via the isolated transmembrane helix still allowed a flexibility regarding rotational and translational motions of the large protein backbone elative to the surface. This led to an effective orientation, enabling solely direct ET of the adsorbed active enzymes. In general, applied potentials revealed reorientations under preservation of the enzyme’s initial binding anchors. In addition, redox changes at the active site of MBH bound on a positively charged biocompatible Au electrode were investigated under turnover conditions as well as under the influence of applied potentials. The second part of this thesis introduced a novel in situ IR spectro-electrochemical approach to monitor protein adsorption on two different transparent conductive oxide (TCO) materials, i.e. antimony-doped tin oxide (ATO) and tin-rich indium oxide (ITOTR). Using horse heart cytochrome c (cyt c) as a model system, protein adsorption on planar ATO and ITOTR thin film electrodes was monitored spectroscopically. Within these studies, a selective protein binding via a histidine affinity tag (His-tag) to the unmodified metal oxide surfaces of ATO and ITOTR was shown. Using the advantage of a controlled immobilization via this affinity tag, Re His-tagged MBH (his-MBH) was directly adsorbed onto ITOTR and also studied in situ by an attenuated total reflection IR (ATR-IR) spectro-electrochemical approach, thereby demonstrating the integrity of the adsorbed protein. Simultaneous PFV studies revealed a remarkable bi-directional activity for H2 cleavage and formation of his-MBH on ITOTR. The latter effect may be related to the specific semiconductor-enzyme interactions, which were discussed in comparison to the unspecific bound Re strep-tagged MBH on the electrode material. Finally, ATR and SEIRA measurements of cyt c and both MBH variants were compared with respect to the signal enhancement, which eventually allowed for an estimation of the catalytic activity of MBH species on different surfaces.Hydrogenasen sind Metalloenzyme, die reversibel die Oxidation von molekularem Wasserstoff in Protonen und Elektronen katalysieren. Das ”Knallgasbakterium“ Ralstonia eutropha H16 (Re) ist eines der wenigen Beispiele, das mehrere [NiFe] Hydrogenasen besitzt, welche Mechanismen der katalytischen H2-Umsetzung unter atmosphärischem Sauerstoffgehalt entwickelt haben. Um das katalytische Potential dieser Sauerstoff-toleranten Hydrogenasen zu erschliessen, insbesondere hinsichtlich möglicher biotechnologischer Anwendungen in biologischen Brennstoffzellen, ist eine effiziente elektronische Kommunikation zwischen Enzym und leitfähiger Oberfläche erforderlich. Dies bedingt ein tiefgreifendes Verständnis der Enzym-Oberflächen-Interaktionen. In dieser Arbeit wurde ein interdisziplinärer Ansatz vorgestellt, der detaillierte Einblicke in die involvierten Adsorptionsparamater der heterodimeren membrangebundenen [NiFe] Hydrogenase (MBH) aus Re auf verschiedenen Elektrodenmaterialien ermöglicht. Der erste Teil dieser Arbeit ist der Adsorptionsstudie der sauerstofftoleranten MBH auf biokompatibel beschichteten Goldelektroden gwidmet. Anhand der Proteinfilm-Voltammetrie (PFV), der Oberflächenverstärkte Infrarot-Absorptions (SEIRA) Spektroelektrochemie Rasterkraftmikroskopie (AFM) und Ellipsometrie wurden die katalytische Aktivität des Enzyms, sowie dessen Struktur, Integrität und Oberflächenbeladung untersucht. Hierfür wurden Resultate der MBH auf geladenen und hydrophoben Oberflächen erhalten. Die Korrelation zwischen der elektrokatalytischen Effizienz und der Art der Oberflächenanbindung des Enzyms auf der Elektrode lieferte wertvolle Informationen zur Orientierung des Enzymes. Die experimentellen Daten wurden durch Molekular-Simulationen und Berechnungen von IR-Spektren mit Hilfe der Dichtefunktionaltheorie (DFT) komplementiert, die in Kooperation durchgeführt wurden. Die vorgeschlagenen Orientierungskonfigurationen, welche auf unterschiedlichen zugänglichen Bindungsankern an der MBH basieren, wurden diskutiert und konnten mittels einer SEIRA-spektroelektrochemischen Studie experimentell nachgewiesen werden. Die so erhaltenen Ergebnisse schlagen insbesondere für eine Immobilizierung der heterodimeren MBH auf leicht negativ geladenen Oberflächen einen optimierten Elektronentransfer an der Enzym-Elektroden-Grenzfläche vor. Hierbei wird eine Immobilisierung der MBH über ihre isolierte Transmembran-Helix angenommen, die über diese Anbindung auch eine Flexibilität des Proteingerüsts hinsichtlich Rotations- als auch Translationsbewegungen relativ zur Oberfläche erlaubt. Dies führt zu einer vorteilhaften Orientierung, die einen direkten Elektronentransfer aller adsorbierten und aktiven Enzyme ermöglicht. Angelegte Elektrodenpotentiale führten teilweise zu Reorientierungen bei Erhalt der ursprünglichen Bindungsanker zwischen Enzym- und Elektrodenoberfläche. Zusätzlich konnten auch Änderungen der Redoxzustände des aktiven Zentrums der MBH unter Turnover-Bedingungen sowie unter dem Einfluss angelegter Potentiale untersucht werden. Der zweite Teil dieser Arbeit führte einen neuen in situ IR spektroelektrochemischen Ansatz zur Beobachtung der Proteinadsorption auf zwei unterschiedlichen, transparenten und leitfähigen Oxidmaterialen (TCO) ein, Antimon-dotiertes Zinn Oxid (ATO) und zinnreiches Indium Oxid (ITOTR). Unter Verwendung von Pferdeherz-Cytochrome c (cyt c) als Modelsystem wurde die Proteinadsorption auf planaren ATO- und ITOTR-Dünnschichtelektroden spektroskopisch verfolgt. In diesem Zusammenhang konnte eine selektive Proteinanbindung über einen Histidin-Affinitätstag (His-tag) an der isolierten Transmembran-Helixauf unmodifizierten Metalloxid-Oberflächen nachgewiesen werden. Diese Art der Anbindung wurde genutzt, um His-getaggte MBH (His-MBH) von Re direkt an die ITOTR Elektroden anzubinden und mittels eines in situ ATR-IR-spektroelektrochemischen Anzatzes zu untersuchen. Hierdurch konnte sowohl der Erhalt der Sekundärstruktur wie auch der strukturellen Integrität des aktiven Zentrums an der Oberfläche demonstriert werden. Simultan ausgeführte PFV-Studien von his-MBH auf ITOTR wiesen eine erstaunliche bi-direktionale Aktivität zur H2-Oxidation und -Reduktion auf. Die ungewöhnliche H2-Reduktion der His-MBH wurde auf spezifische Halbleiter-Enzym-Interaktionen zurückgeführt, welche im Zusammenhang mit der unspezifisch gebundenen streptag MBH auf ITOTR diskutiert wurden. Zum Abschluß wurden Ergebnisse der ATR-IR und SEIRA Spektroskopie von cyt c und beiden MBH Varianten hinsichtlich der Signalverstärkung verglichen und letztendlich eine Abschätzung der katalytischen Aktivität der MBH auf verschiedenen Oberflächen durchgeführt