Electrochemical cofactor recycling of bacterial microcompartments.

Bacterial microcompartments (BMCs) are prokaryotic organelles that consist of a protein shell which sequesters metabolic reactions in its interior. While most of the substrates and products are relatively small and can permeate the shell, many of the encapsulated enzymes require cofactors that must be regenerated inside. We have analyzed the occurrence of an enzyme previously assigned as a cobalamin (vitamin B 12 ) reductase and, curiously, found it in many unrelated BMC types that do not employ B 12 cofactors. We propose NAD+ regeneration as a new function of this enzyme and name it MNdh, for Metabolosome NADH dehydrogenase. Its partner shell protein BMC-T SE assists in passing the generated electrons to the outside. We support this hypothesis with bioinformatic analysis, functional assays, EPR spectroscopy, protein voltammetry and structural modeling verified with X-ray footprinting. This discovery represents a new paradigm for the BMC field, identifying a new, widely occurring route for cofactor recycling and a new function for the shell as separating redox environments.

Creation of a point-of-care therapeutics sensor using protein engineering, electrochemical sensing and electronic integration.

Point-of-care sensors, which are low-cost and user-friendly, play a crucial role in precision medicine by providing quick results for individuals. Here, we transform the conventional glucometer into a 4-hydroxytamoxifen therapeutic biosensor in which 4-hydroxytamoxifen modulates the electrical signal generated by glucose oxidation. To encode the 4-hydroxytamoxifen signal within glucose oxidation, we introduce the ligand-binding domain of estrogen receptor-alpha into pyrroloquinoline quinone-dependent glucose dehydrogenase by constructing and screening a comprehensive protein insertion library. In addition to obtaining 4-hydroxytamoxifen regulatable engineered proteins, these results unveil the significance of both secondary and quaternary protein structures in propagation of conformational signals. By constructing an effective bioelectrochemical interface, we detect 4-hydroxytamoxifen in human blood samples as changes in the electrical signal and use this to develop an electrochemical algorithm to decode the 4-hydroxytamoxifen signal from glucose. To meet the miniaturization and signal amplification requirements for point-of-care use, we harness power from glucose oxidation to create a self-powered sensor. We also amplify the 4-hydroxytamoxifen signal using an organic electrochemical transistor, resulting in milliampere-level signals. Our work demonstrates a broad interdisciplinary approach to create a biosensor that capitalizes on recent innovations in protein engineering, electrochemical sensing, and electrical engineering.

Recent Developments and Applications of Microbial Electrochemical Biosensors.

This chapter provides a comprehensive overview of microbial electrochemical biosensors, which are a unique class of biosensors that utilize the metabolic activity of microorganisms to convert chemical signals into electrical signals. The principles and mechanisms of these biosensors are discussed, including the different types of microorganisms that can be used. The various applications of microbial electrochemical biosensors in fields such as environmental monitoring, medical diagnostics, and food safety are also explored. The chapter concludes with a discussion of future research directions and potential advancements in the field of microbial electrochemical biosensors.

C-H···π interactions disrupt electrostatic interactions between non-aqueous electrolytes to increase solubility.

Grid-scale energy storage applications, such as redox flow batteries, rely on the solubility of redox-active organic molecules. Although redox-active pyridiniums exhibit exceptional persistence in multiple redox states at low potentials (desirable properties for energy storage applications), their solubility in non-aqueous media remains low, and few practical molecular design strategies exist to improve solubility. Here we convey the extent to which discrete, attractive interactions between C-H groups and π electrons of an aromatic ring (C-H···π interactions) can describe the solubility of N-substituted pyridinium salts in a non-aqueous solvent. We find a direct correlation between the number of C-H···π interactions for each pyridinium salt and its solubility in acetonitrile. The correlation presented in this work highlights a consequence of disrupting strong electrostatic interactions with weak dispersion interactions, showing how minimal structural change can dramatically impact pyridinium solubility.

Model-Driven Design of Redox Mediators: Quantifying the Impact of Quinone Structure on Bioelectrocatalytic Activity with Glucose Oxidase.

Successful application of emerging bioelectrocatalysis technologies depends upon an efficient electrochemical interaction between redox enzymes as biocatalysts and conductive electrode surfaces. One approach to establishing such enzyme-electrode interfaces utilizes small redox-active molecules to act as electron mediators between an enzyme-active site and the electrode surface. While redox mediators have been successfully used in bioelectrocatalysis applications ranging from enzymatic electrosynthesis to enzymatic biofuel cells, they are often selected using a guess-and-check approach. Herein, we identify structure-function relationships in redox mediators that describe the bimolecular rate constant for its reaction with a model enzyme, glucose oxidase (GOx). Based on a library of quinone-based redox mediators, a quantitative structure-activity relationship (QSAR) model is developed to describe the importance of mediator redox potential and projected molecular area as two key parameters for predicting the activity of quinone/GOx-based electroenzymatic systems. Additionally, rapid scan stopped-flow spectrophotometry was used to provide fundamental insights into the kinetics and the stoichiometry of reactions between different quinones and the flavin adenine dinucleotide (FAD+/FADH2) cofactor of GOx. This work provides a critical foundation for both designing new enzyme-electrode interfaces and understanding the role that quinone structure plays in altering electron flux in electroenzymatic reactions.

Impact of sodium pyruvate on the electrochemical reduction of NAD+ biomimetics.

Biomimetics of nicotinamide adenine dinucleotide (mNADH) are promising cost-effective alternatives to their natural counterpart for biosynthetic applications; however, attempts to recycle mNADH often rely on coenzymes or precious metal catalysts. Direct electrolysis is an attractive approach for recycling mNADH, but electrochemical reduction of the oxidized mimetic (mNAD+) primarily results in the formation of an enzymatically inactive dimer. Herein, we find that aqueous electrochemical reduction of an NAD+ mimetic, 1-n-butyl-3-carbamoylpyridinium bromide (1+), to its enzymatically active form, 1,4-dihydro-1-n-butyl nicotinamide (1H), is favored in the presence of sodium pyruvate as a supporting electrolyte. Maximum formation of 1H is achieved in the presence of a large excess of pyruvate in combination with a large excess of a co-supporting electrolyte. Formation of 1H is found to be favored at pH 7, with an optimized product ratio of ∼50/50 dimer/1H observed by cyclic voltammetry. Furthermore, sodium pyruvate is shown to promote electroreductive generation of the 1,4-dihydro form of several additional mNADH as well as NADH itself. This method provides a general strategy for regenerating 1,4-dihydro-nicotinamide mimetics of NADH from their oxidized forms.

Establishing a Thermodynamic Landscape for the Active Site of Mo-Dependent Nitrogenase.

Nitrogenase enzymes are the only biological catalysts able to convert N2 to NH3. Molybdenum-dependent nitrogenase consists of two proteins and three metallocofactors that sequentially shuttle eight electrons between three distinct metallocofactors during the turnover of one molecule of N2. While the kinetics of isolated nitrogenase has been extensively studied, little is known about the thermodynamics of its cofactors under catalytically relevant conditions. Here, we employ a recently described pyrene-modified linear poly(ethylenimine) hydrogel to immobilize the catalytic protein of nitrogenase onto an electrode surface. The resulting electroenzymatic interface enabled direct measurement of reduction potentials associated with each metallocofactor of the nitrogenase complex, illuminating the role of nitrogenase reductase in altering the potential landscape in the active site of nitrogenase and revealing the endergonic nature of electron-transfer steps associated with the conversion of N2 to NH3 under physiological conditions.

A synthetic chemist's guide to electroanalytical tools for studying reaction mechanisms.

Monitoring reactive intermediates can provide vital information in the study of synthetic reaction mechanisms, enabling the design of new catalysts and methods. Many synthetic transformations are centred on the alteration of oxidation states, but these redox processes frequently pass through intermediates with short life-times, making their study challenging. A variety of electroanalytical tools can be utilised to investigate these redox-active intermediates: from voltammetry to in situ spectroelectrochemistry and scanning electrochemical microscopy. This perspective provides an overview of these tools, with examples of both electrochemically-initiated processes and monitoring redox-active intermediates formed chemically in solution. The article is designed to introduce synthetic organic and organometallic chemists to electroanalytical techniques and their use in probing key mechanistic questions.

Markov-State Transition Path Analysis of Electrostatic Channeling.

Electrostatic channeling is a naturally occurring approach to control the flux of charged intermediates in catalytic cascades. Computational techniques have enabled quantitative understanding of such mechanisms, augmenting experimental approaches by modeling molecular interactions in atomic detail. In this work, we report the first utilization of a Markov-state model (MSM) to describe the surface diffusion of a reaction intermediate, glucose 6-phosphate, on an artificially modified cascade where hexokinase and glucose-6-phosphate dehydrogenase are covalently conjugated by a cationic oligopeptide bridge. Conformation space networks are used to represent intermediate transport on enzyme surfaces, along with committor probabilities that assess the desorption probability of the intermediate on each segment of the channeling pathway. For the region between the peptide bridge and downstream active site, the ionic strength dependence of desorption probability by MSM agreed well with that by transition state theory. A kinetic Monte Carlo model integrates parameters from different computational methods to evaluate the contribution of desorption during each step. The approach is validated by calculation of kinetic lag time, which agrees well with experimental results. These results further demonstrate the applicability of molecular simulations and advanced sampling techniques to the design of chemical networks.

Bioinspired architecture of a hybrid bifunctional enzymatic/organic electrocatalyst for complete ethanol oxidation.

Electrochemical ethanol oxidation was performed at an innovative hybrid architecture electrode containing TEMPO-modified linear poly(ethylenimine) (LPEI) and oxalate oxidase (OxOx) immobilized on carboxylated multi-walled carbon nanotubes (MWCNT-COOH). On the basis of chromatographic results, the catalytic hybrid electrode system completely oxidized ethanol to CO2 after 12 h of electrolysis. The fact that the developed system can catalyze ethanol electrooxidation at a carbon electrode confirms that organic oxidation catalysts combined with enzymatic catalysts allow up to 12 electrons to be collected per fuel molecule. The Faradaic efficiency of the hybrid system investigated herein lies above 87%. The combination of OxOx with TEMPO-LPEI to obtain a novel hybrid anode to oxidize ethanol to carbon dioxide constitutes a simple methodology with useful application in the development of enzymatic biofuel cells.

Electrochemically Driven, Ni-Catalyzed Aryl Amination: Scope, Mechanism, and Applications.

C-N cross-coupling is one of the most valuable and widespread transformations in organic synthesis. Largely dominated by Pd- and Cu-based catalytic systems, it has proven to be a staple transformation for those in both academia and industry. The current study presents the development and mechanistic understanding of an electrochemically driven, Ni-catalyzed method for achieving this reaction of high strategic importance. Through a series of electrochemical, computational, kinetic, and empirical experiments, the key mechanistic features of this reaction have been unraveled, leading to a second generation set of conditions that is applicable to a broad range of aryl halides and amine nucleophiles including complex examples on oligopeptides, medicinally relevant heterocycles, natural products, and sugars. Full disclosure of the current limitations and procedures for both batch and flow scale-ups (100 g) are also described.

Scalable and safe synthetic organic electroreduction inspired by Li-ion battery chemistry.

Reductive electrosynthesis has faced long-standing challenges in applications to complex organic substrates at scale. Here, we show how decades of research in lithium-ion battery materials, electrolytes, and additives can serve as an inspiration for achieving practically scalable reductive electrosynthetic conditions for the Birch reduction. Specifically, we demonstrate that using a sacrificial anode material (magnesium or aluminum), combined with a cheap, nontoxic, and water-soluble proton source (dimethylurea), and an overcharge protectant inspired by battery technology [tris(pyrrolidino)phosphoramide] can allow for multigram-scale synthesis of pharmaceutically relevant building blocks. We show how these conditions have a very high level of functional-group tolerance relative to classical electrochemical and chemical dissolving-metal reductions. Finally, we demonstrate that the same electrochemical conditions can be applied to other dissolving metal-type reductive transformations, including McMurry couplings, reductive ketone deoxygenations, and epoxide openings.

Investigating the Role of Ligand Electronics on Stabilizing Electrocatalytically Relevant Low-Valent Co(I) Intermediates.

Cobalt complexes have shown great promise as electrocatalysts in applications ranging from hydrogen evolution to C-H functionalization. However, the use of such complexes often requires polydentate, bulky ligands to stabilize the catalytically active Co(I) oxidation state from deleterious disproportionation reactions to enable the desired reactivity. Herein, we describe the use of bidentate electronically asymmetric ligands as an alternative approach to stabilizing transient Co(I) species. Using disproportionation rates of electrochemically generated Co(I) complexes as a model for stability, we measured the relative stability of complexes prepared with a series of N, N-bidentate ligands. While the stability of Co(I)Cl complexes demonstrates a correlation with experimentally measured thermodynamic properties, consistent with an outer-sphere electron transfer process, the set of ligated Co(I)Br complexes evaluated was found to be preferentially stabilized by electronically asymmetric ligands, demonstrating an alternative disproportionation mechanism. These results allow a greater understanding of the fundamental processes involved in the disproportionation of organometallic complexes and have allowed the identification of cobalt complexes that show promise for the development of novel electrocatalytic reactions.

Electrometabolic Pathways: Recent Developments in Bioelectrocatalytic Cascades.

In biological systems, the majority of chemistry occurs in enzymatic pathways. Pathways are essentially cascades of protein catalysts used for catabolism or metabolism. However, in cellular-free systems, catalytic cascades have been rarely studied until recently. This review will introduce the lessons that can be learned from in vivo enzymatic pathways and novel enzymatic pathways that have been developed for synthetic biology of electrochemical energy production and conversion. This review will also discuss the recent bio-inspired developments to utilize catalytic cascades for non-biological applications ranging from energy conversion to biosensing and the electrochemical production of important chemicals such as methanol from carbon dioxide and ammonia from agricultural waste runoff.

Lag Time Spectrophotometric Assay for Studying Transport Limitation in Immobilized Enzymes.

Enzymes are promising catalysts for bioprocessing. For instance, the enzymatic capture of CO2 using carbonic anhydrase (CA) is a carbon capture approach that allows obtaining bicarbonate (HCO3 -) with no high-energy input required. However, application in a commercially viable biotechnology requires sufficient enzymatic lifetime. Although enzyme stabilization can be achieved by different immobilization techniques, most of them are not commercially viable because of transport limitations induced by the immobilization method. Therefore, it is necessary to develop assays for evaluating the role of immobilization on transport limitations. Herein, we describe the development of a fast and reproducible assay for screening immobilized CA by means of absorbance measurement using a computer-controlled microplate reader in stop-flow format. The automated assay allowed minimizing the required volume for analysis to 120 µL. We validated the assay by determining lag times and activities for three immobilization techniques (modified Nafion, hydrogels, and enzyme precipitates), of which linear polyethyleneimine hydrogel showed outstanding performance for CA immobilization.