Electrochemical Trimethylamine N-Oxide Biosensor with Enzyme-Based Oxygen-Scavenging Membrane for Long-Term Operation under Ambient Air

International audienceAn amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O2 reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O2 scavenging membrane. The TMAO sensor operates at a potential of −0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 µM and 15 mM, with a sensitivity of 2.75 ± 1.7 µA/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10% human serum, where the lowest detectable concentration is of 10 µM TMAO

Trimethylamine N ‐Oxide Electrochemical Biosensor with a Chimeric Enzyme

International audienceFor the first time, an enzyme‐based electrochemical biosensor system for determination of trimethylamine N‐oxide (TMAO) is described. It employs an active chimeric variant of TorA in combination with an enzymatically deoxygenating system and a low‐potential mediator for effective regeneration of the enzyme and cathodic current generation. TMAO reductase (TorA) is a molybdoenzyme found in marine and most enterobacteria that specifically catalyzes the reduction of TMAO to trimethylamine (TMA). The chimeric TorA, named TorA‐FDH, corresponds to the apoform of TorA from Escherichia coli reconstituted with the molybdenum cofactor from formate dehydrogenase (FDH). Each enzyme, TorA and TorA‐FDH, was immobilized on the surface of a carbon electrode and protected with a dialysis membrane. The biosensor operates at an applied potential of −0.8 V [vs. Ag/AgCl (1 M KCl)] under ambient air conditions thanks to an additional enzymatic O2‐scavenger system. A comparison between the two enzymatic sensors revealed a much higher sensitivity for the biosensor with immobilized TorA‐FDH. This biosensor exhibits a sensitivity of 14.16 nA/μM TMAO in a useful measuring range of 2–110 μM with a detection limit of LOD=2.96 nM (S/N=3), and was similar for TMAO in buffer and in spiked serum samples. With a response time of 16±2 s, the biosensor is stable over prolonged daily measurements (n=20). This electrochemical biosensor provides suitable applications in detecting TMAO levels in human serum

Structural Determinants of the Catalytic Nia-L Intermediate of [NiFe]-Hydrogenase

[NiFe]-hydrogenases catalyze the reversible cleavage of H2 into two protons and two electrons at the inorganic heterobimetallic NiFe center of the enzyme. Their catalytic cycle involves at least four intermediates, some of which are still under debate. While the core reaction, including H2/H- binding, takes place at the inorganic cofactor, a major challenge lies in identifying those amino acid residues that contribute to the reactivity and how they stabilize (short-lived) intermediate states. Using cryogenic infrared and electron paramagnetic resonance spectroscopy on the regulatory [NiFe]-hydrogenase from Cupriavidus necator, a model enzyme for the analysis of catalytic intermediates, we deciphered the structural basis of the hitherto elusive Nia-L intermediates. We unveiled the protonation states of a proton-accepting glutamate and a Ni-bound cysteine residue in the Nia-L1, Nia-L2, and the hydride-binding Nia-C intermediates, as well as previously unknown conformational changes of amino acid residues in proximity of the bimetallic active site. As such, this study unravels the complexity of the Nia-L intermediate and reveals the importance of the protein scaffold in fine-tuning proton and electron dynamics in [NiFe]-hydrogenase

“Out of Pocket” Protein Binding—A Dilemma of Epitope Imprinted Polymers Revealed for Human Hemoglobin

The epitope imprinting approach applies exposed peptides as templates to synthesize Molecularly Imprinted Polymers (MIPs) for the recognition of the parent protein. While generally the template protein binding to such MIPs is considered to occur via the epitope-shaped cavities, unspecific interactions of the analyte with non-imprinted polymer as well as the detection method used may add to the complexity and interpretation of the target rebinding. To get new insights on the effects governing the rebinding of analytes, we electrosynthesized two epitope-imprinted polymers using the N-terminal pentapeptide VHLTP-amide of human hemoglobin (HbA) as the template. MIPs were prepared either by single-step electrosynthesis of scopoletin/pentapeptide mixtures or electropolymerization was performed after chemisorption of the cysteine extended VHLTP peptide. Rebinding of the target peptide and the parent HbA protein to the MIP nanofilms was quantified by square wave voltammetry using a redox probe gating, surface enhanced infrared absorption spectroscopy, and atomic force microscopy. While binding of the pentapeptide shows large influence of the amino acid sequence, all three methods revealed strong non-specific binding of HbA to both polyscopoletin-based MIPs with even higher affinities than the target peptides.DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"EC/H2020/810856/EU/Twin to Illuminate Metals in Biology and Biocatalysis through Biospectroscopy/TIMB

Hydroxy-bridged Active Site States of [NiFe]-Hydrogenase Unraveled by Cryogenic Vibrational Spectroscopy and DFT Computations

The catalytic mechanism of H2 conversion by [NiFe]-hydrogenase is subject of extensive research. Apart from at least four reaction intermediates of H2/H+ cycling, there is also a number of resting states, which are formed under oxidizing conditions. While not directly involved in the catalytic cycle, knowledge of their molecular structure and reactivity is important, because these states usually accumulate in the course of hydrogenase purification, and they may also play a role in vivo during hydrogenase maturation. Here, we applied low-temperature infrared (cryo-IR) and nuclear resonance vibrational spectroscopy (NRVS) to the isolated catalytic subunit, HoxC, of the heterodimeric regulatory [NiFe]-hydrogenase (RH) from Ralstonia eutropha. Cryo-IR spectroscopy revealed that the HoxC protein can be enriched in almost pure redox states suitable for NRVS investigation. NRVS analysis of the hydrogenase catalytic center is usually hampered by strong spectral contributions of the FeS clusters of the small, electron-transferring subunit. Therefore, our approach to investigate the FeS cluster-free, 57Fe labeled HoxC granted an unprecedented view onto the active site modes, including those obscured by FeS cluster-derived bands. Rationalized by density functional theory (DFT) calculations, our data allow the structural description of two hydroxy-containing resting states. Our work highlights the relevance of cryogenic vibrational spectroscopy and DFT to elucidate the structure of barely defined redox states of the [NiFe]-hydrogenase active site. <br /

Hydroxy-bridged resting states of a [NiFe]-hydrogenase unraveled by cryogenic vibrational spectroscopy and DFT computations

The catalytic mechanism of [NiFe]-hydrogenases is a subject of extensive research. Apart from at least four reaction intermediates of H2/H+ cycling, there are also a number of resting states, which are formed under oxidizing conditions. Although not directly involved in the catalytic cycle, the knowledge of their molecular structures and reactivity is important, because these states usually accumulate in the course of hydrogenase purification and may also play a role in vivo during hydrogenase maturation. Here, we applied low-temperature infrared (cryo-IR) and nuclear resonance vibrational spectroscopy (NRVS) to the isolated catalytic subunit (HoxC) of the heterodimeric regulatory [NiFe]-hydrogenase (RH) from Ralstonia eutropha. Cryo-IR spectroscopy revealed that the HoxC protein can be enriched in almost pure resting redox states suitable for NRVS investigation. NRVS analysis of the hydrogenase catalytic center is usually hampered by strong spectral contributions of the FeS clusters of the small, electron-transferring subunit. Therefore, our approach to investigate the FeS cluster-free, 57Fe-labeled HoxC provided an unprecedented insight into the [NiFe] site modes, revealing their contributions in a spectral range otherwise superimposed by FeS cluster-derived bands. Rationalized by density functional theory (DFT) calculations, our data provide structural descriptions of the previously uncharacterized hydroxy- and water-containing resting states. Our work highlights the relevance of cryogenic vibrational spectroscopy and DFT to elucidate the structure of barely defined redox states of the [NiFe]-hydrogenase active site.DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"EC/H2020/810856/EU/Twin to Illuminate Metals in Biology and Biocatalysis through Biospectroscopy/TIMB