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MATTEO SENSI

Ricercatore Legge 240/10 - t.det.
Dipartimento di Scienze della Vita sede ex Chimica V.Campi 103

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Pubblicazioni

2024 - How Biorecognition Affects the Electronic Properties of Reduced Graphene Oxide in Electrolyte‐Gated Transistor Immunosensors [Articolo su rivista]
Sensi, Matteo; de Oliveira, Rafael Furlan; Berto, Marcello; Paradisi, Alessandro; Greco, Pierpaolo; Bortolotti, Carlo Augusto; Samorì, Paolo; Biscarini, Fabio
abstract

Ambipolar electrolyte-gated transistors (EGTs) based on reduced graphene oxide (rGO) have been demonstrated as ultra-sensitive and highly specific immunosensors. However, the physics and chemistry ruling the device operation are still not fully unraveled. In this work, the aim is to elucidate the nature of the observed sensitivity of the device. Toward this aim, a physical–chemical model that, coupled with the experimental characterization of the rGO-EGT, allows one to quantitatively correlate the biorecognition events at the gate electrode and the electronic properties of rGO-EGT is proposed. The equilibrium of biorecognition occurring at the gate electrode is shown to determine the apparent charge neutrality point (CNP) of the rGO channel. The multiparametric analysis of the experimental transfer characteristics of rGO-EGT reveals that the recognition events modulate the CNP voltage, the excess carrier density Δn, and the quantum capacitance of rGO. This analysis also explains why hole and electron carrier mobilities, interfacial capacitance, the curvature of the transfer curve, and the transconductances are insensitive to the target concentration. The understanding of the mechanisms underlying the transistor transduction of the biorecognition events is key for the interpretation of the response of the rGO-EGT immunosensors and to guide the design of novel and more sensitive devices.

2023 - Reduced Graphene Oxide Electrolyte-Gated Transistor Immunosensor with Highly Selective Multiparametric Detection of Anti-Drug Antibodies [Articolo su rivista]
Sensi, M.; de Oliveira, R. F.; Berto, M.; Palmieri, M.; Ruini, E.; Livio, P. A.; Conti, A.; Pinti, M.; Salvarani, C.; Cossarizza, A.; Cabot, J. M.; Ricart, J.; Casalini, S.; Gonzalez-Garcia, M. B.; Fanjul-Bolado, P.; Bortolotti, C. A.; Samori, P.; Biscarini, F.
abstract

The advent of immunotherapies with biological drugs has revolutionized the treatment of cancers and auto-immune diseases. However, in some patients, the production of anti-drug antibodies (ADAs) hampers the drug efficacy. The concentration of ADAs is typically in the range of 1-10 pm; hence their immunodetection is challenging. ADAs toward Infliximab (IFX), a drug used to treat rheumatoid arthritis and other auto-immune diseases, are focussed. An ambipolar electrolyte-gated transistor (EGT) immunosensor is reported based on a reduced graphene oxide (rGO) channel and IFX bound to the gate electrode as the specific probe. The rGO-EGTs are easy to fabricate and exhibit low voltage operations (& LE; 0.3 V), a robust response within 15 min, and ultra-high sensitivity (10 am limit of detection). A multiparametric analysis of the whole rGO-EGT transfer curves based on the type-I generalized extreme value distribution is proposed. It is demonstrated that it allows to selectively quantify ADAs also in the co-presence of its antagonist tumor necrosis factor alpha (TNF-alpha), the natural circulating target of IFX.

2022 - Monitoring DNA Hybridization with Organic Electrochemical Transistors Functionalized with Polydopamine [Articolo su rivista]
Sensi, M.; Migatti, G.; Beni, V.; D'Alvise, T. M.; Weil, T.; Berto, M.; Greco, P.; Imbriano, C.; Biscarini, F.; Bortolotti, C. A.
abstract

Organic electrochemical transistors (OECTs) are finding widespread application in biosensing, thanks to their high sensitivity, broad dynamic range, and low limit of detection. An OECT biosensor requires the immobilization of a biorecognition probe on the gate, or else on the channel, through several, often lengthy, chemical steps. In this work, a fast and straightforward way to functionalize the carbon gate of a fully screen-printed OECT by means of a polydopamine (PDA) film is presented. By chemical immobilization of an amine-terminated single-stranded oligonucleotide, containing the HSP70 promoter CCAAT sequence, on the PDA film, the detection of the complementary DNA strand is demonstrated. Furthermore, the specificity of the developed genosensor is assessed by comparing its response to the fully complementary strand with the one to partially complementary and noncomplementary oligonucleotides. The developed sensor shows a theoretical limit of detection (LOD) of 100 × 10−15 m and a dynamic range over four orders of magnitude.

2021 - Anti-drug antibody detection with label-free electrolyte-gated organic field-effect transistors [Articolo su rivista]
Sensi, Matteo; Berto, Marcello; Gentile, Sara; Pinti, Marcello; Conti, Andrea; Pellacani, Giovanni; Salvarani, Carlo; Cossarizza, Andrea; Bortolotti, Carlo Augusto; Biscarini, Fabio
abstract

The efficacy of immunotherapy can be undermined by the development of an immune response against a drug/antibody mediated by anti-drug antibodies (ADAs) in treated patients. We present the first label-free EGOFET immunosensor that integrates a biological drug, Nivolumab (Opdivo©), as a specific recognition moiety to quantitatively and selectively detect ADAs against the drug. The limit of detection is 100 fM. This demonstration is a prelude to the detection of ADAs in a clinical setting in the treatment of different pathologies, and it also enables rapid screening of biological drugs for immunogenicity.

2021 - First-Principles Calculations on Ni,Fe-Containing Carbon Monoxide Dehydrogenases Reveal Key Stereoelectronic Features for Binding and Release of CO2to/ from the C-Cluster [Articolo su rivista]
Breglia, R.; Arrigoni, F.; Sensi, M.; Greco, C.; Fantucci, P.; De Gioia, L.; Bruschi, M.
abstract

In view of the depletion of fossil fuel reserves and climatic effects of greenhouse gas emissions, Ni,Fe-containing carbon monoxide dehydrogenase (Ni-CODH) enzymes have attracted increasing interest in recent years for their capability to selectively catalyze the reversible reduction of CO2 to CO (CO2 + 2H+ + 2e- ⇌ CO + H2O). The possibility of converting the greenhouse gas CO2 into useful materials that can be used as synthetic building blocks or, remarkably, as carbon fuels makes Ni-CODH a very promising target for reverse-engineering studies. In this context, in order to provide insights into the chemical principles underlying the biological catalysis of CO2 activation and reduction, quantum mechanics calculations have been carried out in the framework of density functional theory (DFT) on different-sized models of the Ni-CODH active site. With the aim of uncovering which stereoelectronic properties of the active site (known as the C-cluster) are crucial for the efficient binding and release of CO2, different coordination modes of CO2 to different forms and redox states of the C-cluster have been investigated. The results obtained from this study highlight the key role of the protein environment in tuning the reactivity and the geometry of the C-cluster. In particular, the protonation state of His93 is found to be crucial for promoting the binding or the dissociation of CO2. The oxidation state of the C-cluster is also shown to be critical. CO2 binds to Cred2 according to a dissociative mechanism (i.e., CO2 binds to the C-cluster after the release of possible ligands from Feu) when His93 is doubly protonated. CO2 can also bind noncatalytically to Cred1 according to an associative mechanism (i.e., CO2 binding is preceded by the binding of H2O to Feu). Conversely, CO2 dissociates when His93 is singly protonated and the C-cluster is oxidized at least to the Cint redox state.

2021 - Green Fabrication of (6,5)Carbon Nanotube/Protein Transistor Endowed with Specific Recognition [Articolo su rivista]
Berto, M.; Di Giosia, M.; Giordani, M.; Sensi, M.; Valle, F.; Alessandrini, A.; Menozzi, C.; Cantelli, A.; Gazzadi, G. C.; Zerbetto, F.; Calvaresi, M.; Biscarini, F.; Bortolotti, C. A.
abstract

A general single-step approach is introduced for the green fabrication of hybrid biosensors from water dispersion. The resulting device integrates the semiconducting properties of a carbon nanotube (CNT) and the functionality of a protein. In the initial aqueous phase, the protein (viz., lysozyme [LZ]) disperses the (6,5)CNT. Drop-casting of the dispersion on a test pattern (a silicon wafer with interdigitated Au source and drain electrodes) yields a fully operating, robust, electrolyte-gated transistor (EGT) in one step. The EGT response to biorecognition is then assessed using the LZ inhibitor N-acetyl glucosamine trisaccharide. Analysis of the output signal allows one to extract a protein-substrate binding constant in line with values reported for the free (without CNT) system. The methodology is robust, easy to optimize, redirectable toward different targets and sets the grounds for a new class of CNT-protein biosensors that overcome many limitations of the technology of fabrication of CNT biosensors.

2021 - Label free detection of miRNA-21 with electrolyte gated organic field effect transistors (EGOFETs) [Articolo su rivista]
Selvaraj, M.; Greco, P.; Sensi, M.; Saygin, G. D.; Bellassai, N.; D'Agata, R.; Spoto, G.; Biscarini, F.
abstract

We report a dual gate/common channel organic transistor architecture designed for quantifying the concentration of one of the strands of miRNA-21 in solution. The device allows one to measure the differential response between two gate electrodes, viz. one sensing and one reference, both immersed in the electrolyte above the transistor channel. Hybridization with oligonucleotide in the picomolar regime induces a sizable reduction of the current flowing through the transistor channel. The device signal is reported at various gate voltages, showing maximum sensitivity in the sublinear regime, with a limit of detection as low as 35 pM. We describe the dose curves with an analytical function derived from a thermodynamic model of the reaction equilibria relevant in our experiment and device configuration, and we show that the apparent Hill dependence on analyte concentration, whose exponent lies between 0.5 and 1, emerges from the interplay of the different equilibria. The binding free energy characteristic of the hybridization on the device surface is found to be approximately 20% lower with respect to the reaction in solution, hinting to partially inhibiting effect of the surface and presence of competing reactions. Impedance spectroscopy and surface plasmon resonance (SPR) performed on the same oligonucleotide pair were correlated to the electronic current transduced by the EGOFET, and confirmed the selectivity of the biorecognition probe covalently bound on the gold surface.

2021 - Photochemistry and photoinhibition of the H-cluster of FeFe hydrogenases [Articolo su rivista]
Sensi, Matteo; Baffert, Carole; Fourmond, Vincent; De Gioia, Luca; Bertini, Luca; Lèger, Christophe
abstract

Hydrogenases are enzymes that catalyze the oxidation and production of molecular hydrogen. For about fifteen years, there have been many reports about the successful connection of these enzymes to photosensitizers with the aim of designing H2 photoproduction systems, but relatively little attention has been paid to whether and why illumination may affect the catalytic properties of the enzyme. In all hydrogenases, hydrogen activation occurs at an inorganic active site that includes at least one Fe–carbonyl motif, which may make it sensitive to irradiation. Here we review the evidence that hydrogenases are indeed photosensitive. We focus mainly on the so-called FeFe hydrogenases; their active site, called the H-cluster, consists of a [4Fe4S] cluster that is bound by a cysteine sulfur to a diiron site. The iron atoms of the binuclear cluster are coordinated by carbonyl and cyanide ligands and an azadithiolate group. We describe the effects of UV-visible light irradiation on the enzyme under cryogenic or turnover conditions and the photoreactivity of model complexes that mimic the diiron site. We emphasize the dependence of the photochemical processes on wavelength, and warn about FeFe hydrogenase photoinhibition, which should probably be considered when attempts are made to use FeFe hydrogenases for the artificial photosynthesis of solar fuels. We also underline the relevance of studies of synthetic mimics of the H-cluster for understanding at atomistic level the photochemical processes observed in the enzyme.

2021 - Physical insights from the Frumkin isotherm applied to electrolyte gated organic transistors as protein biosensors [Articolo su rivista]
Manco Urbina, Pamela Allison; Berto, Marcello; Greco, Pierpaolo; Sensi, Matteo; Borghi, Simone; Borsari, Marco; Bortolotti, Carlo Augusto; Biscarini, Fabio
abstract

2020 - Flexible Printed Organic Electrochemical Transistors for the Detection of Uric Acid in Artificial Wound Exudate [Articolo su rivista]
Galliani, M.; Diacci, C.; Berto, M.; Sensi, M.; Beni, V.; Berggren, M.; Borsari, M.; Simon, D. T.; Biscarini, F.; Bortolotti, C. A.
abstract

Low-cost, minimally invasive sensors able to provide real-time monitoring of wound infection can enable the optimization of healthcare resources in chronic wounds management. Here, a novel printed organic electrochemical transistors (OECT) biosensor for monitoring uric acid (UA), a bacterial infection biomarker in wounds, is demonstrated in artificial wound exudate. The sensor exploits the enzymatic conversion of UA to 5-hydroxyisourate, catalyzed by Uricase entrapped in a dual-ionic-layer hydrogel membrane casted onto the gate. The sensor response is based on the catalytic oxidation of the hydrogen peroxide, generated as part of the Uricase regeneration process, at the Pt modified gate. The proposed dual membrane avoids the occurrence of nonspecific faradic reactions as, for example, the direct oxidation of UA or other electroactive molecules that would introduce a potentially false negative response. The biosensor is robust and its response is reproducible both in phosphate buffer saline and in complex solutions mimicking the wound exudate. The sensor has a high sensitivity in the range encompassing the pathological levels of UA in wounds (<200 μm) exhibiting a limit of detection of 4.5 μm in artificial wound exudate. All these characteristics make this OECT-based biosensor attractive for wound monitoring interfaced to the patient.

2020 - Harnessing Selectivity and Sensitivity in Electronic Biosensing: A Novel Lab-on-Chip Multigate Organic Transistor [Articolo su rivista]
Parkula, Vitaliy; Berto, Marcello; Diacci, Chiara; Patrahau, Bianca; Di Lauro, Michele; Kovtun, Alessandro; Liscio, Andrea; Sensi, Matteo; Samorì, Paolo; Greco, Pierpaolo; Bortolotti, Carlo A; Biscarini, Fabio
abstract

Electrolyte gated organic transistors can operate as powerful ultrasensitive biosensors, and efforts are currently devoted to devising strategies for reducing the contribution of hardly avoidable, nonspecific interactions to their response, to ultimately harness selectivity in the detection process. We report a novel lab-on-a-chip device integrating a multigate electrolyte gated organic field-effect transistor (EGOFET) with a 6.5 μL microfluidics set up capable to provide an assessment of both the response reproducibility, by enabling measurement in triplicate, and of the device selectivity through the presence of an internal reference electrode. As proof-of-concept, we demonstrate the efficient operation of our pentacene based EGOFET sensing platform through the quantification of tumor necrosis factor alpha with a detection limit as low as 3 pM. Sensing of inflammatory cytokines, which also include TNFα, is of the outmost importance for monitoring a large number of diseases. The multiplexable organic electronic lab-on-chip provides a statistically solid, reliable, and selective response on microliters sample volumes on the minutes time scale, thus matching the relevant key-performance indicators required in point-of-care diagnostics.

2020 - Neuromorphic Organic Devices that Specifically Discriminate Dopamine from Its Metabolites by Nonspecific Interactions [Articolo su rivista]
Giordani, M.; Sensi, M.; Berto, M.; Di Lauro, M.; Bortolotti, C. A.; Gomes, H. L.; Zoli, M.; Zerbetto, F.; Fadiga, L.; Biscarini, F.
abstract

Specific detection of dopamine (DA) is achieved with organic neuromorphic devices with no specific recognition function in an electrolyte solution. The response to voltage pulses consists of amplitude-depressed current spiking mimicking the short-term plasticity (STP) of synapses. An equivalent circuit hints that the STP timescale of the device arises from the capacitance and resistance of the poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) in series with the electrolyte resistance. Both the capacitance and resistance of PEDOT:PSS change with solution compositions. Dose curves are constructed from the STP timescale for each DA metabolite from pM to mM range of concentrations. The STP response of DA is distinctive from the other metabolites even when differences are by one functional group. Both STP and sensitivity to DA are larger across the patho-physiological range with respect to those to DA metabolites. Density functional theory calculations hint to a stronger hydrogen bond pattern of DA ammonium compared to cationic metabolites. The exponential correlation between STP and the binding energy of DA metabolites interacting with PEDOT:PSS indicates that the slow dynamics of ionic species in and out PEDOT:PSS is the origin of the neuromorphic STP. The sensing framework discriminates differences of nonspecific interactions of few kcal mol−1, corresponding to one functional group in the molecule.

2020 - Neuromorphic Organic Devices: Neuromorphic Organic Devices that Specifically Discriminate Dopamine from Its Metabolites by Nonspecific Interactions (Adv. Funct. Mater. 28/2020) [Altro]
Giordani, Martina; Sensi, Matteo; Berto, Marcello; Di Lauro, Michele; Bortolotti, Carlo Augusto; Gomes, Henrique Leonel; Zoli, Michele; Zerbetto, Francesco; Fadiga, Luciano; Biscarini, Fabio
abstract

2019 - Biosensing with Electrolyte Gated Organic Field Effect Transistors [Capitolo/Saggio]
Bortolotti, Carlo Augusto; Berto, Marcello; Sensi, Matteo; DI LAURO, Michele; Biscarini, Fabio
abstract

Electrolyte Gated Organic Field Effect Transistors (EGOFETs) are rapidly emerging as novel players in the field of biosensing: they allow ultra-sensitive, label-free and fast response, and can be employed to sense very diverse analytes, from small molecules to large multimeric proteins. Here, we present the current level of understanding of the working mechanism of EGOFETs, and review some of the most recent and relevant applications as sensors for healthcare and life sciences, discussing advantages and limitations of this technology. EGOFETs appear as a powerful sensing platform that can be readily adapted to the detection of a wide range of biologically relevant species.

2019 - Label free detection of plant viruses with organic transistor biosensors [Articolo su rivista]
Berto, Marcello; Vecchi, Eugenia; Baiamonte, Luca; Condò, Carla; Sensi, Matteo; Di Lauro, Michele; Sola, Marco; De Stradis, Angelo; Biscarini, Fabio; Minafra, Angelantonio; Bortolotti, Carlo Augusto
abstract

Plum Pox Virus (PPV) is the pathogen responsible for Sharka, a highly infectious disease affecting stone fruit trees and causing severe economic damages, which can be only contained through early-detection and frequent monitoring. We propose a bioelectronic PPV biosensor, based on a electrolyte-gated organic field-effect transistor (EGOFET), for the specific detection of PPV in plant extracts with a sub ng/ml detection limit. The sensing unit of the biosensor is based on anti-PPV antibodies, uniformly oriented on the gold gate electrode by using a sub-monolayer of Protein G. The sensitivity and dynamic range of the EGOFET-based biosensor are comparable to those of commercially available platforms for detection of plant pathogens. This novel electronic immunosensor is compatible with low-cost fabrication procedures and can be easily reconfigured into a fully portable device to be operated in greenhouse and in the field orchards.

2019 - Modulating the Faradic Operation of All-Printed Organic Electrochemical Transistors by Facile in Situ Modification of the Gate Electrode [Articolo su rivista]
Sensi, Matteo; Berto, Marcello; Candini, Andrea; Liscio, Andrea; Cossarizza, Andrea; Beni, Valerio; Biscarini, Fabio; Bortolotti, Carlo Augusto
abstract

Organic electrochemical transistors (OECTs) operated in the faradic regime were shown as outperforming transducers of bioelectric signals in vitro and in vivo. Fabrication by additive manufacturing techniques fosters OECTs as ideal candidates for point-of-care applications, as well as imposes limitations on the choice of materials and their processing conditions. Here, we address the question of how the response of fully printed OECTs depends on gate electrode material. Toward this end, we investigate the redox processes underlying the operation of OECTs under faradic regime, to show OECTs with carbon gate (C-gate) that exhibit no current modulation gate voltages <1.2 V. This is a hallmark that no interference with the faradic operation of the device enabled by redox processes occurs when operating C-gate OECTs in the low-voltage range as label-free biosensors for the detection of electroactive (bio)molecules. To tune the faradic response of the device, we electrodeposited Au on the carbon gate (Au-C-gate), obtaining a device that operates at lower gate voltage values than C-gate OECT. The presence of gold on the gate allowed further modification of the electrical performances by functionalization of the Au-C-gate with different self-assembled monolayers by fast potential-pulse-assisted method. Moreover, we show that the presence in the electrolyte solution of an external redox probe can be used to drive the faradic response of both C- and Au-C-gate OECTs, impacting on the gate potential window that yields effective drain current modulation. The results presented here suggest possible new strategies for controlling the faradic operation regime of OECTs sensors by chemical modification of the gate surface.

2018 - Electrochemical Investigations of Hydrogenases and Other Enzymes That Produce and Use Solar Fuels [Articolo su rivista]
Del Barrio, M.; Sensi, M.; Orain, C.; Baffert, C.; Dementin, S.; Fourmond, V.; Leger, C.
abstract

Conspectus Many enzymes that produce or transform small molecules such as O2, H2, and CO2 embed inorganic cofactors based on transition metals. Their active site, where the chemical reaction occurs, is buried in and protected by the protein matrix, and connected to the solvent in several ways: chains of redox cofactors mediate long-range electron transfer; static or dynamic tunnels guide the substrate, product and inhibitors; amino acids and water molecules transfer protons. The catalytic mechanism of these enzymes is therefore delocalized over the protein and involves many different steps, some of which determine the response of the enzyme under conditions of stress (extreme redox conditions, presence of inhibitors, light), the catalytic rates in the two directions of the reaction and their ratio (the "catalytic bias"). Understanding all the steps in the catalytic cycle, including those that occur on sites of the protein that are remote from the active site, requires a combination of biochemical, structural, spectroscopic, theoretical, and kinetic methods. Here we argue that kinetics should be used to the fullest extent, by extracting quantitative information from the comparison of data and kinetic models and by exploring the combination of experimental kinetics and theoretical chemistry. In studies of these catalytic mechanisms, direct electrochemistry, the technique which we use and contribute to develop, has become unescapable. It simply consists in monitoring the changes in activity of an enzyme that is wired to an electrode by recording an electric current. We have described kinetic models that can be used to make sense of these data and to learn about various aspects of the mechanism that are difficult to probe using more conventional methods: long-range electron transfer, diffusion along gas channels, redox-driven (in)activations, active site chemistry and photoreactivity under conditions of turnover. In this Account, we highlight a few results that illustrate our approach. We describe how electrochemistry can be used to monitor substrate and inhibitor diffusion along the gas channels of hydrogenases and we discuss how the kinetics of intramolecular diffusion relates to global properties such as resistance to oxygen and catalytic bias. The kinetics and/or thermodynamics of intramolecular electron transfer may also affect the catalytic bias, the catalytic potentials on either side of the equilibrium potential, and the overpotentials for catalysis (defined as the difference between the catalytic potentials and the open circuit potential). This is understood by modeling the shape of the steady-state catalytic response of the enzyme. Other determinants of the catalytic rate, such as domain motions, have been probed by examining the transient catalytic response recorded at fast scan rates. Last, we show that combining electrochemical investigations and MD, DFT, and TD-DFT calculations is an original way of probing the reactivity of the H-cluster of hydrogenase, in particular its reactions with CO, O2, and light. This approach contrasts with the usual strategy which aims at stabilizing species that are presumed to be catalytic intermediates, and determining their structure using spectroscopic or structural methods.

2018 - Experimental and theoretical study of the reaction of FeFe hydrogenases with dioxygen [Articolo su rivista]
Orain, C.; Sensi, M.; Baffert, C.; Fourmond, V.; Leger, C.
abstract

Hydrogenases are enzymes that catalyze hydrogen oxidation and production. The so-called "FeFe hydrogenases", the active site of which is a [Fe6(CN)2(CO)3] cluster, are particularly efficient. Their inhibition by O2 prevents them from being used for H2 production. Combining electrochemical experiments, site-directed mutagenesis, molecular dynamics calculations and quantum chemistry calculations allowed to elucidate all steps of the reaction with O2 and to predict the rate of inhibition. These results will pave the way for engineering enzymes that resist O2.

2018 - Interaction of the H-Cluster of FeFe Hydrogenase with Halides [Articolo su rivista]
Del Barrio, M.; Sensi, M.; Fradale, L.; Bruschi, M.; Greco, C.; De Gioia, L.; Bertini, L.; Fourmond, V.; Leger, C.
abstract

FeFe hydrogenases catalyze H2 oxidation and production using an "H-cluster", where two Fe ions are bound by an aza-dithiolate (adt) ligand. Various hypotheses have been proposed (by us and others) to explain that the enzyme reversibly inactivates under oxidizing, anaerobic conditions: intramolecular binding of the N atom of adt, formation of the so-called "Hox/inact" state or nonproductive binding of H2 to isomers of the H-cluster. Here, we show that none of the above explains the new finding that the anaerobic, oxidative, H2-dependent reversible inactivation is strictly dependent on the presence of Cl- or Br-. We provide experimental evidence that chloride uncompetitively inhibits the enzyme: it reversibly binds to catalytic intermediates of H2 oxidation (but not to the resting "Hox" state), after which oxidation locks the active site into a stable, saturated, inactive form, the structure of which is proposed here based on DFT calculations. The halides interact with the amine group of the H-cluster but do not directly bind to iron. It should be possible to stabilize the inhibited state in amounts compatible with spectroscopic investigations to explore further this unexpected reactivity of the H-cluster of hydrogenase.

2017 - Mechanism of O2 diffusion and reduction in FeFe hydrogenases [Articolo su rivista]
Kubas, A.; Orain, C.; De Sancho, D.; Saujet, L.; Sensi, M.; Gauquelin, C.; Meynial-Salles, I.; Soucaille, P.; Bottin, H.; Baffert, C.; Fourmond, V.; Best, R. B.; Blumberger, J.; Leger, C.
abstract

FeFe hydrogenases are the most efficient H2-producing enzymes. However, inactivation by O2 remains an obstacle that prevents them being used in many biotechnological devices. Here, we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We propose that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of a highly reactive OH radical and hydroxylated cysteine. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species during prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.

2017 - New perspectives in hydrogenase direct electrochemistry [Articolo su rivista]
Sensi, M.; del Barrio, M.; Baffert, C.; Fourmond, V.; Leger, C.
abstract

Electrochemical studies of hydrogenases, the biological catalysts of H2 oxidation and production, have proven wrong the old saying that enzymes do not easily transfer electrons to electrodes in the absence of mediators. Many distinct hydrogenases have actually been directly connected to electrodes or particles, for studying their catalytic mechanism or for designing solar-fuels catalysts. In this review, we list the electrodes that have proved successful for direct electron transfer to hydrogenases, and we discuss recent results which illustrate new directions in this research field: the study of the biosynthesis of FeFe hydrogenase, the electrochemical characterization of non-standard NiFe or FeFe hydrogenases, the general discussion of what makes a catalyst better in one particular direction of the reaction, and the elucidation of the molecular mechanisms of hydrogenase catalysis by combining electrochemistry and theoretical chemistry, spectroscopy or photochemistry. The electrochemical methods described herein will probably prove useful for studying or using other redox enzymes.

2017 - Photoinhibition of FeFe hydrogenase [Articolo su rivista]
Sensi, M.; Baffert, C.; Fradale, L.; Gauquelin, C.; Soucaille, P.; Meynial-Salles, I.; Bottin, H.; De Gioia, L.; Bruschi, M.; Fourmond, V.; Leger, C.; Bertini, L.
abstract

In the enzyme FeFe hydrogenase, hydrogen oxidation and production occur at the H-cluster, a Fe6S6 active site that bears intrinsic carbonyl and cyanide ligands. This enzyme has been coupled to photosensitizers to design H2 photoproduction systems, and yet, according to earlier reports, the enzyme from Desulfovibrio desulfuricans is "easily destroyed" in "normal laboratory light". Here we report direct electrochemistry measurements of the effect of light on the activity of the enzymes from Chlamydomonas reinhardtii and Clostridium acetobutylicum, together with TDDFT and DFT calculations of the reactivity of the excited states of the H-cluster. We conclude that visible light does not inhibit these enzymes, but absorption of UVB (280-315 nm) irreversibly damages the H-cluster by triggering the release of an intrinsic CO ligand; the resulting unsaturated species rearranges and protonates to form a stable, inactive dead-end. Answering the question of which particular hydrogenase can resist which particular wavelengths is important regarding solar H2 production, and our results show that some but not all FeFe hydrogenases can actually be combined with photosensitizers that utilize the solar spectrum, provided a UV screen is used. We suggest that further investigations of the compatibility of hydrogenases or hydrogenase mimics with lightharvesting systems should also consider the possibility of irreversible photoinhibition.

2016 - Reactivity of the Excited States of the H-Cluster of FeFe Hydrogenases [Articolo su rivista]
Sensi, M.; Baffert, C.; Greco, C.; Caserta, G.; Gauquelin, C.; Saujet, L.; Fontecave, M.; Roy, S.; Artero, V.; Soucaille, P.; Meynial-Salles, I.; Bottin, H.; De Gioia, L.; Fourmond, V.; Leger, C.; Bertini, L.
abstract

FeFe hydrogenases catalyze H2 oxidation and formation at an inorganic active site (the "H-cluster"), which consists of a [Fe2(CO)3(CN)2(dithiomethylamine)] subcluster covalently attached to a Fe4S4 subcluster. This active site is photosensitive: visible light has been shown to induce the release of exogenous CO (a reversible inhibitor of the enzyme), shuffle the intrinsic CO ligands, and even destroy the H-cluster. These reactions must be understood because they may negatively impact the use of hydrogenase for the photoproduction of H2. Here, we explore in great detail the reactivity of the excited states of the H-cluster under catalytic conditions by examining, both experimentally and using TDDFT calculations, the simplest photochemical reaction: the binding and release of exogenous CO. A simple dyad model can be used to predict which excitations are active. This strategy could be used for probing other aspects of the photoreactivity of the H-cluster.