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Direct Electrochemistry of Redox Enzymes As a Tool for Mechanistic Studies Christophe Léger, Patrick Bertrand

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Direct Electrochemistry of Redox Enzymes As a Tool for Mechanistic Studies Christophe Léger, Patrick Bertrand Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies Christophe Léger, Patrick Bertrand To cite this version: Christophe Léger, Patrick Bertrand. Direct Electrochemistry of Redox Enzymes as a Tool for Mech- anistic Studies. Chemical Reviews, American Chemical Society, 2007, 108, 10.1021/cr0680742. hal- 01211439 HAL Id: hal-01211439 https://hal.archives-ouvertes.fr/hal-01211439 Submitted on 5 Oct 2015 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Chem. Rev. 2008, 108, 2379–2438 2379 Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies Christophe Le´ger* and Patrick Bertrand Laboratoire de Bioe´nerge´tique et Inge´nierie des Prote´ines, CNRS UPR 9036, IBSM, and Universite´ de Provence, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France Received August 1, 2007 Contents 5. List of Abbreviations 2432 6. Acknowledgments 2432 1. Introduction 2379 7. Supporting Information Available 2432 2. Modeling the Voltammetry of Adsorbed Redox 2382 8. References 2432 Enzymes 2.1. Noncatalytic Voltammetry 2382 1. Introduction 2.1.1. The Case of Pure Electron Transfer (ET) 2382 2.1.2. Chemical Processes Coupled to ET 2387 This review regards the use of dynamic electrochemistry 2.2. Catalytic Voltammetry in the Absence of Mass 2388 to study the mechanism of redox enzymes, with exclusive Transport Control emphasis on the configuration where the protein is adsorbed 2.2.1. Transition from Noncatalytic to Pure 2388 onto an electrode and electron tranfer is direct. We still often Catalytic Regimes come across the statement these days that redox enzymes 2.2.2. From Phenomenological Equations to 2389 are too large and too fragile to interact directly with a metallic Kinetic Models electrode without being at least partly denatured. It is still 2.2.3. The Basic Model 2390 held that since the active site of these enzymes is deeply 2.2.4. Substrate Binding and Release 2391 buried in the protective protein matrix, direct electron 2.2.5. Slow Interfacial ET Kinetics 2393 exchange with an electrode can only occur under exceptional conditions. Yet 20 years have passed since it was shown 2.2.6. Intramolecular ET Kinetics 2397 that direct electron transfer (ET) can occur between an 2.2.7. Redox Activation and Inactivation 2398 electrode and a large, catalytically active enzyme,1–13 and 2.3. Effect of Mass Transport 2399 about one hundred examples have already been reported. The 2.3.1. Rotating Disk Electrode 2400 oxidoreductases that are most auspicious for achieving direct 2.3.2. Stationary Electrode 2401 “wiring” interact with their soluble redox partner (cyto- 2.4. Chronoamperometry at the Rotating Disk 2402 chrome, ferredoxin, quinone, or redox dye) by an outer Electrode sphere ET which occurs at (or close to) the surface of the 2.4.1. Stepwise Changes in Reactants 2403 enzyme, where the electrode can also interact. Having redox Concentration enzymes directly connected to electrodes made it possible 2.4.2. Continuous Changes in Reactants 2403 to exploit the naturally high efficiency of these biological Concentration systemsfordevelopingselectivethird-generationbiosensors,14–16 3. Case Studies 2404 environmentally sound biofuel cells,17-19 heterogeneous 3.1. Prelims 2404 catalysts,20 and even biomolecular electronic components.21 3.1.1. Attaching the Enzyme 2404 By combining dynamic electrochemistry and scanning probe 3.1.2. Observing Reasonable Noncatalytic 2407 microscopic techniques, it has now become possible to Signals characterize the protein-electrode interface and electron 3.1.3. The Right Reactions, at a Reasonable 2408 exchange processes in great detail.22–25 Closer to our Potential and Overpotential concerns, this configuration has recently proved useful for 3.1.4. The Right Michaelis Parameters and Their 2409 learning about the kinetic and molecular aspects of the Dependences on pH catalytic mechanism of redox enzymessthis is the topic of 3.1.5. Catalytic Signal Being Independent of 2410 the present review. Protein/Electrode Interactions Redox enzymes intervene in a number of biological 3.2. Interfacial and Intermolecular Electron 2411 processes, the most important of which may be the bioen- Transfer ergetic metabolism.26 In living organisms, many essential 3.3. Intramolecular Electron Transfer (iET) 2413 reactions are thermodynamically allowed only because they 3.4. Active Site Chemistry 2418 are coupled to the very exergonic hydrolysis of ATP. The 3.5. Bidirectional Catalysis 2425 nonredox enzyme called ATP-synthase is responsible for 3.6. Redox-Dependent, Slow or Irreversible 2427 maintaining the ATP/ADP ratio far from equilibrium by (In)activation Processes catalyzing the endergonic phosphorylation of ADP; as a 3.7. Substrate Channels 2430 source of free energy, this enzyme uses a gradient of 4. Conclusion 2432 electrochemical potential of the proton, which is built by the redox enzymes in the respiratory and photosynthetic * E-mail: [email protected]. chains, hence the importance of electron-transfer reactions 10.1021/cr0680742 CCC: $71.00 2008 American Chemical Society Published on Web 07/11/2008 2380 Chemical Reviews, 2008, Vol. 108, No. 7 Le´ger and Bertrand doreductase) is a transmembrane complex assembled from 45 different proteins with a total molecular weight approach- ing 1 MDa; it houses a flavin at the site of NADH oxidation and nine iron-sulfur (FeS) clusters.28–30 Most interestingly, an enzyme does much more than catalyzing a reaction: it does so at the right rate, considering its substrate is often the product of another enzyme and Vice Versa; it resists inactivation by potent inhibitors and stressful chemicals present in the cell; it fastens partly-transformed catalytic intermediates that may be harmful, etc. Hence, the enzymes’ structures and mechanisms are complex because their catalytic properties are manifold. A significant difference between the chemist’s synthetic inorganic catalysts and the large multicenter enzymes we Christophe Le´ger obtained his Ph.D. in physical chemistry at the University study is that the mechanism of the latter involves a number of Bordeaux I, France, where he studied dendritic patterning in elec- of steps of various nature, which occur in a concerted manner trodeposition under the supervision of Franc¸oise Argoul. In 1999, he joined on distinct sites of the protein that may be very far apart Fraser Armstrong at the University of Oxford, U.K., to study redox enzymes from one another. We illustrate this by considering the by electrochemistry, and he has been fascinated by this topic ever since. mechanism of NiFe hydrogenase,31 whose structure is shown In 2002, he was hired by the French National Center for Scientific Research in Figure 1. This enzyme catalyzes the reversible transforma- (CNRS) in Marseilles. He is interested in bioinorganic chemistry, in enzyme tion between molecular hydrogen and protons according to kinetics, and in studying the mechanism of multicenter redox enzymes. + - H2 a 2H + 2e . It is a complex of two proteins and consists of about 10000 atoms, including one Ni and 12 Fe ions. The active site chemistry per se, involving the hetero- lytic splitting of dihydrogen, occurs at a NiFe cluster which is buried inside the protein matrix. The electrons produced or consumed at the active site are sequentially transferred to the redox partner (a cytochrome) via a chain of FeS clusters. The active site is also connected to the solvent by gas channels and by a yet unidentified proton-transfer machinery, involving protonable amino acid side chains and ordered water molecules. Some of the steps in the mecha- nism, e.g. proton and electron transfers and diffusion along the channels, remain difficult to study using conventional techniques, and very little is known about how the interplay between all these events gives the enzyme its global properties in terms of selectivity, catalytic directionality, and Patrick Bertrand received his undergraduate education at the Ecole resistance to chemical stress. Centrale de Paris. After having worked for three years as scientific attache´ at the French Embassy in Bucharest, he joined the Laboratory of Over the last 15 years, the elucidation of the three- Condensed Matter Electronics at the University of Provence, in Marseilles, dimensional structures of many redox enzymes (and par- where he started to study the magnetic properties of iron-sulfur clusters ticularly metalloenzymes) has inspired research on their under the supervision of Jean-Pierre Gayda. He received his Ph.D. in mechanism by providing a molecular framework for inter- physics in 1977 and his doctorat es sciences in 1981. Since 1989, he preting their biochemical and spectroscopic properties, and has been a Professor at the Universite´ de Provence. In 1993, he participated with his group in the creation of the Laboratory of Bioenergetics an instrumental road map for studies based on genetic and Engineering of Proteins of the CNRS, headed by Mireille Bruschi. engineering. A major contemporary challenge is to decipher Although he enjoyed
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