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

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

Photoelectrochemistry of Photosystem II in Vitro vs in Vivo.

Factors governing the photoelectrochemical output of photosynthetic microorganisms are poorly understood, and energy loss may occur due to inefficient electron transfer (ET) processes. Here, we systematically compare the photoelectrochemistry of photosystem II (PSII) protein-films to cyanobacteria biofilms to derive: (i) the losses in light-to-charge conversion efficiencies, (ii) gains in photocatalytic longevity, and (iii) insights into the ET mechanism at the biofilm interface. This study was enabled by the use of hierarchically structured electrodes, which could be tailored for high/stable loadings of PSII core complexes and Synechocystis sp. PCC 6803 cells. The mediated photocurrent densities generated by the biofilm were 2 orders of magnitude lower than those of the protein-film. This was partly attributed to a lower photocatalyst loading as the rate of mediated electron extraction from PSII in vitro is only double that of PSII in vivo. On the other hand, the biofilm exhibited much greater longevity (>5 days) than the protein-film (<6 h), with turnover numbers surpassing those of the protein-film after 2 days. The mechanism of biofilm electrogenesis is suggested to involve an intracellular redox mediator, which is released during light irradiation

An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14.

Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1-2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays, and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. The differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.The work presented in this paper was supported by grants from the BBSRC: BB/G016240/1 BBSRC Sustainable Energy Centre Cell Wall Sugars Programme (BSBEC) and grant BB/K005537/1. JCM’s work at the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE -AC02-05CH11231. NFB was supported by a PhD studentship from the Portuguese Foundation for Science and Technology. AN was supported by a summer studentship award from the Biochemical Society. The authors are grateful to the European Community’s Seventh Framework Programme SUNLIBB (FP7/2007-2013) under the grant agreement no 251132.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/tpj.1289

The Development of Biophotovoltaic Systems for Power Generation and Biological Analysis.

Biophotovoltaic systems (BPVs) resemble microbial fuel cells, but utilise oxygenic photosynthetic microorganisms associated with an anode to generate an extracellular electrical current, which is stimulated by illumination. Study and exploitation of BPVs have come a long way over the last few decades, having benefited from several generations of electrode development and improvements in wiring schemes. Power densities of up to 0.5 W m-2 and the powering of small electrical devices such as a digital clock have been reported. Improvements in standardisation have meant that this biophotoelectrochemical phenomenon can be further exploited to address biological questions relating to the organisms. Here, we aim to provide both biologists and electrochemists with a review of the progress of BPV development with a focus on biological materials, electrode design and interfacial wiring considerations, and propose steps for driving the field forward

A Si photocathode protected and activated with a Ti and Ni composite film for solar hydrogen production.

An efficient, stable and scalable hybrid photoelectrode for visible-light-driven H2 generation in an aqueous pH 9.2 electrolyte solution is reported. The photocathode consists of a p-type Si substrate layered with a Ti and Ni-containing composite film, which acts as both a protection and electrocatalyst layer on the Si substrate. The film is prepared by the simple drop casting of the molecular single-source precursor, [{Ti2(OEt)9(NiCl)}2] (TiNipre), onto the p-Si surface at room temperature, followed by cathodic in situ activation to form the catalytically active TiNi film (TiNicat). The p-Si|TiNicat photocathode exhibits prolonged hydrogen generation with a stable photocurrent of approximately -5 mA cm(-2) at 0 V vs. RHE in an aqueous pH 9.2 borate solution for several hours, and serves as a benchmark non-noble photocathode for solar H2 evolution that operates efficiently under neutral-alkaline conditions.This is the final published version of a paper published in Chemistry - A European Journal Volume 21, Issue 10, pages 3919–3923, March 2, 2015, DOI: 10.1002/chem.20140656

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

A non-hybrid method for the PDF equations of turbulent flows on unstructured grids

In probability density function (PDF) methods of turbulent flows, the joint PDF of several flow variables is computed by numerically integrating a system of stochastic differential equations for Lagrangian particles. A set of parallel algorithms is proposed to provide an efficient solution of the PDF transport equation, modeling the joint PDF of turbulent velocity, frequency and concentration of a passive scalar in geometrically complex configurations. An unstructured Eulerian grid is employed to extract Eulerian statistics, to solve for quantities represented at fixed locations of the domain (e.g. the mean pressure) and to track particles. All three aspects regarding the grid make use of the finite element method (FEM) employing the simplest linear FEM shape functions. To model the small-scale mixing of the transported scalar, the interaction by exchange with the conditional mean model is adopted. An adaptive algorithm that computes the velocity-conditioned scalar mean is proposed that homogenizes the statistical error over the sample space with no assumption on the shape of the underlying velocity PDF. Compared to other hybrid particle-in-cell approaches for the PDF equations, the current methodology is consistent without the need for consistency conditions. The algorithm is tested by computing the dispersion of passive scalars released from concentrated sources in two different turbulent flows: the fully developed turbulent channel flow and a street canyon (or cavity) flow. Algorithmic details on estimating conditional and unconditional statistics, particle tracking and particle-number control are presented in detail. Relevant aspects of performance and parallelism on cache-based shared memory machines are discussed.Comment: Accepted in Journal of Computational Physics, Feb. 20, 200