Electrocatalysis by heme enzymes—applications in biosensing

Funding Information: The APC was funded by TIMB3 project, European Union's Horizon 2020 Research and Innovation Program grant agreement No 810856. Funding Information: Acknowledgments: We acknowledge the support from Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE 2020-Programa Operacional Competitividade e Internacionalização (POCI); from FCT—Fundação para a Ciência e a Tecnologia (PTDC/BIA-BFS/31026/2017 and 2020.05017.BD) and from the European Union's Horizon 2020 Research and Innovation Program, through TIMB3 and B-LigZymes projects (grant agreements No 810856 and 824017, respectively). We thank Edilson Galdino for critical reading of the manuscript and helpful discussions. Funding Information: Funding: The APC was funded by TIMB3 project, European Union's Horizon 2020 Research and Innovation Program grant agreement No 810856. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.Heme proteins take part in a number of fundamental biological processes, including oxygen transport and storage, electron transfer, catalysis and signal transduction. The redox chemistry of the heme iron and the biochemical diversity of heme proteins have led to the development of a plethora of biotechnological applications. This work focuses on biosensing devices based on heme proteins, in which they are electronically coupled to an electrode and their activity is determined through the measurement of catalytic currents in the presence of substrate, i.e., the target analyte of the biosensor. After an overview of the main concepts of amperometric biosensors, we address transduction schemes, protein immobilization strategies, and the performance of devices that explore reactions of heme biocatalysts, including peroxidase, cytochrome P450, catalase, nitrite reductase, cytochrome c oxidase, cytochrome c and derived microperoxidases, hemoglobin, and myoglobin. We further discuss how structural information about immobilized heme proteins can lead to rational design of biosensing devices, ensuring insights into their efficiency and long-term stability.publishersversionpublishe

Etude des relations structure-fonction dans des protéines photosynthétiques par spectroscopie différentielle dans les domaines IR moyen et lointain

Les métalloprotéines jouent un rôle central dans diverses voies métaboliques, couvrant une grande variété de fonctions par la diversité des cations métalliques, des sphères de coordination et des structures spécifiques des centres métalliques. Cette thèse de doctorat porte sur l’étude des relations structure - fonction de différentes métalloprotéines de la cyanobactérie Thermosynechoccocus elongatus impliquées dans la photosynthèse, par spectroscopie infra rouge dans les domaines moyen et lointain, couplée soit à l’électrochimie, soit à la photochimie. Les propriétés d'une ferredoxine mineure Fd2 ont été comparées à celles de la ferredoxine principale Fd1. Différents potentiels d’oxydoréduction (Em) ont été obtenus pour Fd1 et Fd2. Des différences significatives ont été observées pour Fd2, notamment concernant les interactions entre le centre [2Fe-2S] et la protéine, révélées par le mode (Fe-St) identifié dans le domaine IR lointain. Les propriétés de PsbVII et Tll0287 ont été comparées à celles d'un mutant, M58C. Le Em de PsbV2 a été déterminé ainsi que sa dépendance en pH ainsi que celle de M58C. L'importance de l'environnement de l’hème dans la modulation des propriétés redox a été mise en évidence par spectroscopie IRTF en mode différentiel. Un nouveau marqueur de la coordination axiale His/Cys est proposé entre 289 cm-1 et 303 cm-1, dont la fréquence pourrait être corrélée à la valeur du Em. L’analyse de la transition S1 à S2 dans le PSII a impliqué la mise au point du dispositif de mesure. L'étude d'échantillons H2O, D2O et H218O et de PSII avec du Sr incorporé de façon biosynthétique a permis d’identifier des modes du centre Mn4CaO5 et de molécules d’eau ligands.Metalloproteins play a central role in various metabolic pathways of living organisms, covering a large variety of functions by the diversity of the metal cations, coordination spheres, and specific structures of the metal centres. This PhD thesis concerns the structure-function relationship of different metalloproteins of the cyanobacterium Thermosynechoccocus elongatus involved in photosynthesis, using spectroscopic methods, notably Mid- to Far- IR spectroscopy, coupled to either electrochemistry or photochemistry. The properties of a minor ferredoxin Fd2 were compared with those of the main ferredoxin Fd1. Different midpoint potentials were obtained for Fd1 and the newly purified Fd2. Different interactions between the [2Fe-2S] cluster and the protein scaffold, were evidenced by the Amide modes as well as the Far-IR (Fe-St) mode. The properties of PsbVII and Tll0287 were compared to those of a mutant (M58C). The oxidation-reduction potential of PsbV2 was determined as well as its pH dependence and that of M58C. With Tll0287, these His/Cys c-type cytochromes show a very negative Em, which differs significantly from one cytochrome to another. The importance of the heme environment in modulating redox properties was demonstrated by FTIR difference spectroscopy. A new His/Cys axial coordination marker is proposed between 289 cm-1 and 303 cm-1, whose frequency could be correlated with the Em value. The analysis of the S1 to S2 transition in the PSII involved the adaptation of a measurement device. The study of H2O, D2O and H218O samples and PSII with biosynthetically incorporated Sr allowed the identification of IR modes of the Mn4CaO5 centre and ligand water molecules

Molecular Details on Multiple Cofactor Containing Redox Metalloproteins Revealed by Infrared and Resonance Raman Spectroscopies

Vibrational spectroscopy and in particular, resonance Raman (RR) spectroscopy, can provide molecular details on metalloproteins containing multiple cofactors, which are often challenging for other spectroscopies. Due to distinct spectroscopic fingerprints, RR spectroscopy has a unique capacity to monitor simultaneously and independently different metal cofactors that can have particular roles in metalloproteins. These include e.g., (i) different types of hemes, for instance hemes c, a and a3 in caa3-type oxygen reductases, (ii) distinct spin populations, such as electron transfer (ET) low-spin (LS) and catalytic high-spin (HS) hemes in nitrite reductases, (iii) different types of Fe-S clusters, such as 3Fe-4S and 4Fe-4S centers in di-cluster ferredoxins, and (iv) bi-metallic center and ET Fe-S clusters in hydrogenases. IR spectroscopy can provide unmatched molecular details on specific enzymes like hydrogenases that possess catalytic centers coordinated by CO and CN− ligands, which exhibit spectrally well separated IR bands. This article reviews the work on metalloproteins for which vibrational spectroscopy has ensured advances in understanding structural and mechanistic properties, including multiple heme-containing proteins, such as nitrite reductases that house a notable total of 28 hemes in a functional unit, respiratory chain complexes, and hydrogenases that carry out the most fundamental functions in cells.DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"EC/H2020/810856/EU/Twin to Illuminate Metals in Biology and Biocatalysis through Biospectroscopy/TIMB

Professional exposure to basaltic rock dust: Assessment by the Vibrio fischeri ecotoxicological test

A recent study demonstrates that inhalation of airborne particulate from Mount Etna eruptions may induce fibrotic lung disease. The occupational exposure of construction workers from the Etna area, who excavate building sites and use basalt dust to make mortar, has never been assessed

SERR Spectroelectrochemistry as a Guide for Rational Design of DyP-Based Bioelectronics Devices

Immobilised dye-decolorizing peroxidases (DyPs) are promising biocatalysts for the development of biotechnological devices such as biosensors for the detection of H2O2. To this end, these enzymes have to preserve native, solution properties upon immobilisation on the electrode surface. In this work, DyPs from Cellulomonas bogoriensis (CboDyP), Streptomyces coelicolor (ScoDyP) and Thermobifida fusca (TfuDyP) are immobilised on biocompatible silver electrodes functionalized with alkanethiols. Their structural, redox and catalytic properties upon immobilisation are evaluated by surface-enhanced resonance Raman (SERR) spectroelectrochemistry and cyclic voltammetry. Among the studied electrode/DyP constructs, only CboDyP shows preserved native structure upon attachment to the electrode. However, a comparison of the redox potentials of the enzyme in solution and immobilised states reveals a large discrepancy, and the enzyme shows no electrocatalytic activity in the presence of H2O2. While some immobilised DyPs outperform existing peroxidase-based biosensors, others fail to fulfil the essential requirements that guarantee their applicability in the immobilised state. The capacity of SERR spectroelectrochemistry for fast screening of the performance of immobilised heme enzymes places it in the front-line of experimental approaches that can advance the search for promising DyP candidates

Protonation of the Cysteine Axial Ligand Investigated in His/Cys c -Type Cytochrome by UV–Vis and Mid- and Far-IR Spectroscopy

International audienceHis/Cys coordination was recently found in several c-type cytochromes, which could act as sensors, in electron transport or in regulation. Towards better understanding Cys function and reactivity in these cytochromes, we compare cytochrome c6 (c6wt) from the cyanobacterium Nostoc PCC 7120 with its Met58Cys mutant. We probe the axial ligands and heme properties by combining visible and Mid to Far FTIR difference spectroscopies. Cys58 determines the strong negative redox potential and pH dependence of M58C (EmM58C = -375 mV, versus Emc6wt = +339 mV). Mid-IR (notably Cys ν(SH), His ν(C5N1), heme δ(CmH)) and Far-IR (ν(Fe(II)-His), ν(His-Fe(III)-Cys)) markers of the heme and ligands show that Cys58 remains a strong thiolate ligand of reduced Met58Cys at alkaline pH, while it is protonated at pH 7.5, stabilized by a strong hydrogen bonding interaction, and weakly interacts with Fe(II). These data provide a benchmark for further analysis of c-type cytochromes with natural His/Cys coordination

Unusual structures and unknown roles of FeS clusters in metalloenzymes seen from a resonance Raman spectroscopic perspective

Funding Information: This work was financially supported by: Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionaliza??o (POCI) and by national funds through FCT - Funda?a?o para a Cie?ncia e a Tecnologia; by the European Union's Horizon 2020 research and innovation programme, TIMB3, GA No 810856. ST acknowledges PTDC/BTM-SAL/29507/2017 and CMS acknowledges PTDC/BIA-BFS/31026/2017 project and CB acknowledges 2020.05017.BD fellowship granted by FCT. GC, IZ and PH thank the Einstein Foundation Berlin (grant number EVF-2016-277) for funding. This work was also supported through the cluster of excellence ?UniSysCat? funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany?s Excellence Strategy-EXC2008/1-390540038 and the SPP 1927 ?Iron sulfur for Life? - ZE 510/2-2 (IZ). Funding Information: This work was financially supported by: Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT - Fundação para a Ciência e a Tecnologia; by the European Union’s Horizon 2020 research and innovation programme, TIMB 3 , GA No 810856. ST acknowledges PTDC/BTM-SAL/29507/2017 and CMS acknowledges PTDC/BIA-BFS/31026/2017 project and CB acknowledges 2020.05017.BD fellowship granted by FCT. GC, IZ and PH thank the Einstein Foundation Berlin (grant number EVF-2016-277) for funding. This work was also supported through the cluster of excellence “UniSysCat“ funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germanýs Excellence Strategy-EXC2008/1-390540038 and the SPP 1927 ”Iron sulfur for Life” - ZE 510/2-2 (IZ). Publisher Copyright: © 2021 The AuthorsThe universe of known biological FeS clusters is constantly enlarging. Besides the conventional, well described [2Fe–2S], [3Fe–4S] and cubane [4Fe–4S] clusters, novel, unprecedented structures are emerging. They include unusually coordinated clusters, with additional sulfur atoms, e.g., [4Fe–5S], [5Fe–5S], [4Fe–4S]-5S-[4Fe–4S], [8Fe–7S], [8Fe–9S] and [8Fe–8S–C] and heteronuclear clusters, e.g., [Ni–4Fe–4S], [2Ni–4Fe–4S], [4Fe–4S]-[2Ru], [Me–7Fe–9S–C–(homocitrate)] that undertake versatile physiological roles in the activation of small molecules (H2, CO2, CO and N2) and in the sulfuration of different compounds (e.g., t-RNAs, biotin and lipoic acid) in biology. A few structures are characterized by highly distorted geometries, e.g., the non-cubane [4Fe–4S] center and the hydrogenase-related [4Fe–3S] cluster, and contain atypical ligations or vacant coordination sites, which confer them novel functions far from the common electron transfer. Herein, we single out clusters found in i) hydrogenases that ensure sustainable hydrogen cycling, promising a clean fuel production in the future, ii) radical-SAM enzymes that can inspire applied catalysis due to an intrinsic flexibility of the radical chemistry, and iii) standard [4Fe–4S] cluster with still unknown function in DNA repair enzymes, which offer a possibility to interfere with DNA repair in pathogens or improve it in humans. Focusing on the abovementioned enzymes, we demonstrate the unique power of resonance Raman spectroscopy to unveil remarkable features in FeS centers, which has contributed to our understanding of unusual structures and disentangling of unknown functions.publishersversionpublishe

An alternative plant-like cyanobacterial ferredoxin with unprecedented structural and functional properties: Ferredoxin with low Em discriminating against FNR

International audiencePhotosynthetic [2Fe-2S] plant-type ferredoxins have a central role in electron transfer between the photosynthetic chain and various metabolic pathways. Several genes are coding for [2Fe2S] ferredoxins in cyanobacteria, with four in the thermophilic cyanobacterium Thermosynechococcus elongatus. The structure and functional properties of the major ferredoxin Fd1 are well known but data on the other ferredoxins are scarce. We report the structural and functional properties of a novel minor type ferredoxin, Fd2 of T. elongatus, homologous to Fed4 from Synechocystis sp. PCC 6803. Remarkably, the midpoint potential of Fd2, Em = -440 mV, is lower than that of Fd1, Em = -372 mV. However, while Fd2 can efficiently react with photosystem I or nitrite reductase, time-resolved spectroscopy shows that Fd2 has a very low capacity to reduce ferredoxin-NADP+ oxidoreductase (FNR). These unique Fd2 properties are discussed in relation with its structure, solved at 1.38 Å resolution. The Fd2 structure significantly differs from other known ferredoxins structures in loop 2, N-terminal region, hydrogen bonding networks and surface charge distributions. UV-Vis, EPR, and Mid- and Far-IR data also show that the electronic properties of the [2Fe2S] cluster of Fd2 and its interaction with the protein differ from those of Fd1 both in the oxidized and reduced states. The structural analysis allows to propose that valine in the motif Cys53ValAsnCys56 of Fd2 and the specific orientation of Phe72, explain the electron transfer properties of Fd2. Strikingly, the nature of these residues correlates with different phylogenetic groups of cyanobacterial Fds. With its low redox potential and its discrimination against FNR, Fd2 exhibits a unique capacity to direct efficiently photosynthetic electrons to metabolic pathways not dependent on FNR