DOCSLIB.ORG

Characterization of an Electron Conduit Between Bacteria and the Extracellular Environment

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Characterization of an Electron Conduit Between Bacteria and the Extracellular Environment Characterization of an electron conduit between bacteria and the extracellular environment Robert S. Hartshornea, Catherine L. Reardonb, Daniel Rossc, Jochen Nuesterd, Thomas A. Clarkea, Andrew J. Gatesa, Paul C. Millsa, Jim K. Fredricksonb, John M. Zacharab, Liang Shib, Alex S. Beliaevb, Matthew J. Marshallb, Ming Tienc, Susan Brantleyd, Julea N. Butta, and David J. Richardsona,1 aSchool of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom; bPacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352; and Departments of cBiochemistry and Molecular Biology, and dGeoscience, Pennsylvania State University, University Park, PA 16802-4500 Edited by Dianne Newman, Massachusetts Institute of Technology, Cambridge, MA, and accepted by the Editorial Board September 9, 2009 (received for review January 7, 2009) A number of species of Gram-negative bacteria can use insoluble transfer across the OM is unclear, because its amino acid sequence minerals of Fe(III) and Mn(IV) as extracellular respiratory electron does not display a canonical redox cofactor binding motif (Fig. S1). acceptors. In some species of Shewanella, deca-heme electron MtrA is predicted to be a deca-heme periplasmic protein (Fig. S1) transfer proteins lie at the extracellular face of the outer mem- (10), which associates with the OM as part of an MtrCAB complex brane (OM), where they can interact with insoluble substrates. To (5). Because the OM is Ϸ40 Å wide, and MtrA and MtrC are reduce extracellular substrates, these redox proteins must be predicted to be on opposite sides of it, it is not immediately obvious charged by the inner membrane/periplasmic electron transfer how electrons can pass between them in vivo. This study presents system. Here, we present a spectro-potentiometric characteriza- a characterization of the MtrCAB complex that leads us the tion of a trans-OM icosa-heme complex, MtrCAB, and demonstrate propose a novel trans-OM electron transfer system, in which the its capacity to move electrons across a lipid bilayer after incorpo- MtrB porin serves as a sheath within which MtrA and MtrC can ration into proteoliposomes. We also show that a stable MtrAB embed sufficiently at the inner and outer faces of the membrane, subcomplex can assemble in the absence of MtrC; an MtrBC respectively, for electron transfer to take place between them. subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We Results and Discussion BIOCHEMISTRY propose a model for the modular organization of the MtrCAB Spectral Characterization of the MtrCAB Complex in Micelles and complex in which MtrC is an extracellular element that mediates Liposome Membranes. The MtrCAB complex was purified from cell electron transfer to extracellular substrates and MtrB is a trans-OM membranes of Shewanella oneidensis solubilized in Triton X-100 spanning ␤-barrel protein that serves as a sheath, within which (TX100) (Fig. S2). SDS/PAGE revealed that two subunits stained MtrA and MtrC exchange electrons. We have identified the MtrAB for heme-dependent peroxidase activity (Fig. 1A), which were module in a range of bacterial phyla, suggesting that it is widely confirmed as MtrC (Ϸ85 kDa) and MtrA (Ϸ40 kDa) by probing used in electron exchange with the extracellular environment. with specific antibodies. An additional band running slightly slower than MtrC was also visualized on gels stained with Coomassie blue cytochrome-c ͉ iron respiration ͉ protein film voltammetry ͉ (Fig. 1B). Cross-reactivity toward MtrB antibody and analysis by electron paramagnetic resonance ͉ Shewanella MS confirmed that this protein was the OM ␤-barrel porin MtrB. Analysis of the MtrCAB preparations by sedimentation equilib- ␮ number of species of Gram-negative bacteria can couple rium (SE) at a range of concentrations (0.25–2.5 M) and rotation Aanaerobic growth to the respiratory reduction of mineral- rates (7, 9, and 11 krpm) confirmed that the protein behaved as a oxides, such as those of Fe(III) and Mn(IV), that are insoluble in single homogenous species with an apparent molecular mass app Ϸ water at neutral pH and so unable to enter the cell (1). Energy (Mw )of 210 kDa (Fig. 2A), consistent with a 1:1:1 ratio of conservation in Gram-negative bacteria relies on the generation of MtrCAB in a heterotrimeric icosa-heme complex (6). The solution a proton-motive force across the inner membrane (IM). Electrons UV-visible spectrum of MtrCAB in TX100 micelles is typical of low ␥ reduce the IM quinone (Q) pool to quinol (QH ) via the activity of spin c-type cytochromes with absorption maxima at 410 nm ( ) and 2 ␥ ␤ proton-motive enzymes, such as formate dehydrogenase or hydro- 540 nm in the oxidized state and 422 nm ( ), 520 nm ( ), and 552 nm (␣) in the reduced state (Fig. 3A). To assess whether MtrCAB genase (1). In order for continued turnover, the QH2 pool needs to be reoxidized. For water soluble electron acceptors, the systems can catalyze transmembrane electron transfer, the complex was that perform this function are associated with the IM or periplasm, incorporated into liposomes with the redox dye methylviologen where access to the active sites of these enzymes is straightforward. (MV) entrapped within the vesicles. In control experiments, it was established that addition of dithionite (up to 170 ␮M) to the However, cycling the QH2 pool is a problem when the available electron acceptor cannot enter the cell, as in the case of Fe(III) MV-liposomes in the absence of the MtrCAB complex did not minerals. One of the electron-transfer models proposed for extra- result in the reduction of the internalized MV (Fig. S3A). However, cellular Fe(III) reduction in Shewanella species implicates deca- when the MV-liposomes were solublized using TX100 reduction of heme c-type cytochromes encoded by the omcA-mtrCAB gene the MV did occur as indicated by the development of the reduced Ϸ Ϸ cluster (2–5). MtrC and OmcA are located on the extracellular face peaks at 395 and 600 nm (Fig. S3A). When MV-proteolipo- of the outer membrane (OM), where they can be directly involved in electron transfer to extracellular Fe(III) minerals (6, 7), or to Author contributions: R.S.H., C.L.R., T.A.C., J.K.F., J.M.Z., L.S., A.S.B., M.J.M., M.T., S.B., extracellular electron shuttles, which in turn reduce Fe(III) minerals J.N.B., and D.J.R. designed research; R.S.H., C.L.R., D.R., J.N., T.A.C., A.J.G., P.C.M., L.S., not physically associated with the cell (8, 9). Importantly, electron A.S.B., and M.J.M. performed research; R.S.H., C.L.R., D.R., J.N., T.A.C., A.J.G., and J.N.B. delivery to the extracellular deca-heme cytochromes requires that analyzed data; and T.A.C., J.K.F., J.M.Z., M.T., S.B., J.N.B., and D.J.R. wrote the paper. electrons originating from the IM are transferred across the OM. The authors declare no conflict of interest. The biochemical basis for this electron transfer is not understood. This article is a PNAS Direct Submission. D.N. is a guest editor invited by the Editorial Board. MtrB is predicted to be an OM ␤-barrel porin comprising 28 1To whom correspondence should be addressed. E-mail: [email protected]. ␤ transmembrane -strands that is critical for extracellular Fe(III) This article contains supporting information online at www.pnas.org/cgi/content/full/ respiration (10). However, the role performed by MtrB in electron 0900086106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900086106 PNAS ͉ December 29, 2009 ͉ vol. 106 ͉ no. 52 ͉ 22169–22174 Downloaded by guest on September 29, 2021 A the spectral features of reduced MV also developed when dithionite B was added to the MtrCAB MV-proteolipsomes (see red spectrum in Fig. 3B). These features are partially obscured by the reduced MtrCAB absorption spectrum and the light scattering from the liposomes; however, the sharp 395-nm absorption band and broad Ϸ600-nm absorption bands characteristic of reduced MV could be clearly resolved in the reduced minus oxidized difference spectra of the proteoliposomes (see red line in Fig. 3B Inset), and were absent from the reduced minus oxidized difference spectrum of the MtrCAB in solution (see black line in Fig. 3B Inset). Control experiments were carried out in which proteoliposomes were ultracentrifuged after treatment with dithionite. The supernatants were collected and reduced with dithionite. No signals character- istic of reduced MV were apparent (Fig. S3D), which confirmed that the proteoliposomes did not leak MV after this treatment. C The liposome experiments demonstrate that at least some of the MtrCAB molecules reconstitute into MV-proteoliposomes in a topology that allows them to mediate electron transfer between externally added dithionite and the entrapped MV, the function- ality of the complex in transmembrane electron transfer. Addition of Fe(III) citrate to the dithionite-reduced MV-proteoliposomes resulted in reoxidation of the MtrCAB complex as judged by the ␥ Fig. 1. SDS/PAGE analysis of Mtr proteins. (A and B) Two replica samples were shift of the -band from 422 to 410 nm and disappearance of the resolved by 12% (wt/vol) SDS/PAGE and stained for heme-dependent perox- 552 nm peak (see green spectrum in Fig. 3B). Reoxidation of idase activity (A) or with Coomassie blue (B). Lane 1, molecular mass markers the internalized reduced MV also occurred as indicated by the (from top of gel: 100, 75, 50, 37.5, 25, and 15 kDa); lane 2, MtrCAB; lane 3, disappearance of the 395 and 600 nm absorption bands of reduced MtrAB.
Load more

Recommended publications
  • Electro-Organic Reactions and Redox Active Biomolecules: a Student Diary*
    these techniques. Some group members also told me that they use liquid chromatography-mass spectrometry (LC- MS), and it is a great help in analyzing their reaction products. Methods like atomic force microscopy, scanning electron microscopy, and surface FTIR are used in the group for characterization of the electrode surfaces they make. A week or so later: I have done my first electrochemical synthesis experiment. I did cyclic voltammetry Electro-organic Reactions in undergraduate analytical lab, so it was not totally new. The reactor cell I used was like two connected beakers in a water jacket (Fig. 1). One beaker is and Redox Active Biomolecules: the working electrode compartment, and the other is the counter electrode compartment, separated by a salt bridge. A Student Diary* This is so that products from the two compartments do not combine to give by James F. Rusling and Albert J. Fry undesirable products. The reactor had a carbon cloth working electrode, which is a cool material, like a conducting It is early December, and I have been in chemistry grad school black fabric that you cut to the right for about 3 months. It is a big transition, and a bit tougher size with scissors. It has a large surface than I expected. The courses are not too hard, but the focus area to facilitate the catalytic reaction. The reactor has counter and reference here is very much on research. I have chosen the group in electrodes, and is hooked up to a which I will do my thesis research, and I am happy about that.
    [Show full text]
  • Application of Voltammetry in Biomedicine-Recent
    Macedonian Journal of Chemistry and Chemical Engineering, Vol. 39, No. 2, pp. 153–166 (2020) MJCCA9 – 803 ISSN 1857-5552 e-ISSN 1857-5625 Received: September 23, 2020 DOI: 10.20450/mjcce.2020.2152 Accepted: October 8, 2020 Original scientific paper APPLICATION OF VOLTAMMETRY IN BIOMEDICINE RECENT ACHIEVEMENTS IN ENZYMATIC VOLTAMMETRY Rubin Gulaboski1, Valentin Mirceski2,3 1Faculty of Medical Sciences, Goce Delčev University, Štip, Republic of Macedonia 2Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University, Skopje, Republic of Macedonia 3Department of Inorganic and Analytical Chemistry, University of Lodz, Tamka 12, 91-403 Lodź, Poland [email protected]; [email protected] Protein-film voltammetry (PFV) is considered the simplest methodology to study the electrochem- istry of lipophilic redox enzymes in an aqueous environment. By anchoring particular redox enzymes on the working electrode surface, it is possible to get an insight into the mechanism of enzyme action. The PFV methodology enables access to the relevant thermodynamic and kinetic parameters of the enzyme- electrode reaction and enzyme-substrate interactions, which is important to better understand many meta- bolic pathways in living systems and to delineate the physiological role of enzymes. PFV additionally provides important information which is useful for designing specific biosensors, simple medical devices and bio-fuel cells. In the current review, we focus on some recent achievements of PFV, while presenting some novel protocols that contribute to a better communication between redox enzymes and the working electrode. Insights to several new theoretical models that provide a simple strategy for studying electrode reactions of immobilized enzymes and that enable both kinetic and thermodynamic characterization of enzyme-substrate interactions are also provided.
    [Show full text]
  • Electrochemical Evidence That Pyranopterin Redox Chemistry Controls the Catalysis of Yedy, a Mononuclear Mo Enzyme
    Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY, a mononuclear Mo enzyme Hope Adamsona, Alexandr N. Simonovb, Michelina Kierzekc, Richard A. Rotheryc, Joel H. Weinerc, Alan M. Bondb, and Alison Parkina,1 aDepartment of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom; bSchool of Chemistry, Monash University, Clayton, VIC 3800, Australia; and cDepartment of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved October 13, 2015 (received for review August 25, 2015) A long-standing contradiction in the field of mononuclear Mo explores the possibility that ligand-based redox chemistry plays a enzyme research is that small-molecule chemistry on active-site mimic role in YedY catalysis. compounds predicts ligand participation in the electron transfer YedY has been structurally characterized via both X-ray crys- reactions, but biochemical measurements only suggest metal-cen- tallography and X-ray absorption spectroscopy (XAS) (3, 8, 9). In tered catalytic electron transfer. With the simultaneous measurement most mononuclear Mo enzymes, heme groups and iron sulfur of substrate turnover and reversible electron transfer that is provided clusters are found within the same protein as the Mo center, but the by Fourier-transformed alternating-current voltammetry, we show only metal site in YedY is Mo, making this enzyme a helpfully that Escherichia coli YedY is a mononuclear Mo enzyme that recon- simple system for studying redox chemistry (Fig. 1) (1, 3). Within ciles this conflict. In YedY, addition of three protons and three elec- the active site, the X-ray structure was interpreted to show Mo(V) trons to the well-characterized “as-isolated” Mo(V) oxidation state is in a square pyramidal environment (3), identical to other members needed to initiate the catalytic reduction of either dimethyl sulfoxide of the “sulfite oxidase” family of mononuclear Mo enzymes.
    [Show full text]
  • Electrochemical Properties of Gsnos by Protein Film Voltammetry
    180 Chapter 6 Electrochemical Properties of gsNOS by Protein Film Voltammetry 181 6.1 Abstract The accurate measurement of a protein’s electrochemical properties is an important part of understanding its function. Several methods have been developed to facilitate communication between deeply buried protein metal centers and electrodes. One such technique, protein film voltammetry (PFV), involves the immobilization of proteins on the surface of electrodes by various means. Such techniques can result in clear signals from proteins, allowing the measurement of not only reduction potentials but kinetics as well. Two types of PFV have been employed in the study of the nitric oxide sythase from Geobacillus stearothermophilus. First, a mutant of this NOS was covalently connected to a gold electrode. It was hypothesized that the use of a hydrophilic linker would maintain a normal aqueous environment around the enzyme and avoid the shifting of potentials (a common problem in PFV). When it was found that this technique still resulted in measuring responses with significantly shifted potentials (as compared with those measured by redox titration in solution), a more traditional film was employed. The kinetics of gsNOS was studied in DDAB films and compared with the mammalian inducible isoform. It was found to show similar behavior, and experiments are still underway to further characterize the kinetics of wild type and three mutants of gsNOS (W70H, W70F, and W70Y, introduced in Chapter 3). 182 6.2 Introduction and Background Key to the function of most metalloenzymes is the redox activity of transition metals. These metals have the unique ability, as distinct from most organic molecules, to easily access multiple oxidation states.
    [Show full text]
  • 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.
    [Show full text]
  • AC Fields 339, 346, 351 Adsorption – Enzymes 14, 250 – Nonspecific 45
    389 Index a biocathodes 243ff. AC fi elds 339, 346, 351 – air diffusion 252 adsorption – hydrogel-based 252 – enzymes 14, 250 – oxygen reduction reaction (ORR) 243ff. – nonspecifi c 45, 217 – polarization plots 251 – proteins 13f., 174 biocompatible 12, 16, 42ff. – reactivity 111 bioelectrocatalysis, see biocatalysis – surface-specifi c 111 biofuel cell (BFC) 3, 229ff. – vesicle 197f. – development 6, 26 alcohol dehydrogenase 260f. – electron transfer (ET) 29ff. alkanethiols 98, 100 – enzymatic glucose/O2 38 amperometric biosensors 1ff. – enzyme-based, see enzymatic fuel cell anesthesia 377f., 380 (EFC) antibodies 3, 53f. – principle 31 antigen 3, 53f. biofuel cell 2f., 5f., 9f., 29, 31f., 36ff., 55, ascorbate oxidase 244 57 assembled biofuel cells 255 bioinorganic hybrids 85 atomic force microscopy (AFM) 85, 92 biological recognition element 1ff., 7, 16, – contact mode 287 23f., 29, 36, 38, 44ff., 56f., 59 – fl uidity of biomimetic membranes 200f. biological recognition 4, 11 – small unilamellar vesicles 198 – element 27, 45 – TERS 291 biomimetic layers 107f., 143f., 183, 190 atomic force microscopy 85 – fl uidity 200f. Au atom mining 104 – formation of lipid fi lms 194ff. Au–nanoparticle hybrids 120 – gold-supported 201 AuNP–azurin hybrid 123 – rapid solvent exchange 200 AuNP–protein hybrids 123 – vesicle fusion 196ff. azurin 113f., 123, 125 biomimetic membrane 189ff., 200f., 220 biosensor architecture 2f., 5, 13ff., 18ff., b 38f., 55f., 59 bending rigidity 338, 347 biosensor stability 28, 43 between laboratory precision, see – long-term 4, 6, 16, 28, 44 reproducibility – storage 28 bias voltage 92, 95, 97 – working 28 bilayer lipid membranes 107 biosensor bilayer lipid membranes, see membranes – accuracy 28 bilirubin oxidase 244, 246 – amperometric 1ff.
    [Show full text]
  • Probing Biological Redox Chemistry with Large Amplitude Fourier Transformed Ac Voltammetry Chemcomm
    Volume 53 Number 69 7 September 2017 Pages 9507–9650 ChemComm Chemical Communications rsc.li/chemcomm ISSN 1359-7345 FEATURE ARTICLE Alan M. Bond, Alison Parkin et al. Probing biological redox chemistry with large amplitude Fourier transformed ac voltammetry ChemComm View Article Online FEATURE ARTICLE View Journal | View Issue Probing biological redox chemistry with large amplitude Fourier transformed ac voltammetry Cite this: Chem. Commun., 2017, 53, 9519 Hope Adamson,a Alan M. Bond*b and Alison Parkin *a Biological electron-exchange reactions are fundamental to life on earth. Redox reactions underpin Received 18th May 2017, respiration, photosynthesis, molecular biosynthesis, cell signalling and protein folding. Chemical, Accepted 12th June 2017 biomedical and future energy technology developments are also inspired by these natural electron transfer DOI: 10.1039/c7cc03870d processes. Further developments in techniques and data analysis are required to gain a deeper understanding of the redox biochemistry processes that power Nature. This review outlines the new insights gained from rsc.li/chemcomm developing Fourier transformed ac voltammetry as a tool for protein film electrochemistry. Creative Commons Attribution 3.0 Unported Licence. Introduction Biological redox reactions are mediated by both small and large molecules, ranging from species such as quinones, which resemble typical synthetic chemistry redox reagents, to complex metalloenzymes comprising ‘‘wires’’ of redox-active centers (i.e. cytochrome c oxidase, the enzyme responsible for converting O2 1 to H2O in the mitochondria of all human cells, see Fig. 1). Redox reactions even underpin some DNA repair mechanisms.2,3 This This article is licensed under a review focuses on redox active proteins and enzymes, and the new mechanistic insights which can be gained by integrating the technique of large amplitude Fourier transformed voltammetry4 (FTacV) into the toolbox of protein film electrochemistry.
    [Show full text]
  • Relation Between Anaerobic Inactivation and Oxygen Tolerance in a Large Series of Nife Hydrogenase Mutants
    Relation between anaerobic inactivation and oxygen tolerance in a large series of NiFe hydrogenase mutants Abbas Abou Hamdana, Pierre-Pol Liebgottb, Vincent Fourmonda, Oscar Gutiérrez-Sanzc, Antonio L. De Laceyc, Pascale Infossia, Marc Rousseta, Sébastien Dementina, and Christophe Légera,1 aLaboratoire de Bioénergétique et Ingénierie des Protéines, Centre National de la Recherche Scientifique, Aix-Marseille Université, Institut de Microbiologie de la Méditerranée, 13402 Marseille Cedex 20, France; bLaboratoire de Microbiologie, Institut de Recherche pour le Développement, Aix Marseille Université, Unité Mixte de Recherche D180, Microbiologie et Biotechnologie des Environnements Chauds, Ecole Supérieure d’Ingénieurs de Luminy, 13288 Marseille Cedex 09, France; and cLaboratorio de Bioelectrocatalisis, Departamento de Biocatalisis, Instituto de Catalisis y Petroleoquimica, Consejo Superior de Investigaciones Cientificas, 28049 Madrid, Spain Edited* by Jean-Michel Savéant, Université Paris Diderot (Paris 7), Paris, France, and approved October 19, 2012 (received for review July 29, 2012) Nickel-containing hydrogenases, the biological catalysts of H2 investigations have shown that in O2-resistant enzymes, it is an oxidation and production, reversibly inactivate under anaerobic, unprecedented 4Fe3S cluster (8–10) that has a high reduction oxidizing conditions. We aim at understanding the mechanism of potential and the ability to cycle through three redox states in (in)activation and what determines its kinetics, because there is a narrow range of potential (11). a correlation between fast reductive reactivation and oxygen tol- The rate of reactivation of NiB partly determines O2 toler- erance, a property of some hydrogenases that is very desirable ance, but the reason this reaction is orders of magnitude faster in oxygen-resistant hydrogenases than in standard hydrogenases from the point of view of biotechnology.
    [Show full text]
  • Membrane Protein Modified Electrodes in Bioelectrocatalysis
    catalysts Review Membrane Protein Modified Electrodes in Bioelectrocatalysis Huijie Zhang y, Rosa Catania y and Lars J. C. Jeuken * School of Biomedical Sciences and the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK; [email protected] (H.Z.); [email protected] (R.C.) * Correspondence: [email protected]; Tel.: +44-113-343-3829 These authors have contributed equally. y Received: 18 November 2020; Accepted: 3 December 2020; Published: 6 December 2020 Abstract: Transmembrane proteins involved in metabolic redox reactions and photosynthesis catalyse a plethora of key energy-conversion processes and are thus of great interest for bioelectrocatalysis- based applications. The development of membrane protein modified electrodes has made it possible to efficiently exchange electrons between proteins and electrodes, allowing mechanistic studies and potentially applications in biofuels generation and energy conversion. Here, we summarise the most common electrode modification and their characterisation techniques for membrane proteins involved in biofuels conversion and semi-artificial photosynthesis. We discuss the challenges of applications of membrane protein modified electrodes for bioelectrocatalysis and comment on emerging methods and future directions, including recent advances in membrane protein reconstitution strategies and the development of microbial electrosynthesis and whole-cell semi-artificial photosynthesis. Keywords: membrane protein; bioelectrocatalysis; electrode modification; biofuel cells; photosynthesis; liposomes; hybrid vesicles; microbial electrosynthesis 1. Introduction Membrane proteins constitute 20–30% of all proteins encoded by both prokaryotic and eukaryotic cells. They perform a wide variety of functions, including material transport, signal transduction, catalysis, proton and electron transport (Figure1)[ 1]. They are also key to a number of earth’s most fundamental reactions, such as respiration and photosynthesis [2,3].
    [Show full text]
  • Protein Film Voltammetry
    View Article Online / Journal Homepage / Table of Contents for this issue Reactions of complex metalloproteins studied by protein-film voltammetry Fraser A. Armstrong, Hendrik A. Heering and Judy Hirst Inorganic Chemistry Laboratory, Oxford University, Oxford, UK OX1 3QR The following review explores applications of voltammetric systems. The approach we will describe stems from the original methods for observing reactions of complex metalloproteins. discoveries of Hill and of Kuwana and their coworkers1.2who Attention is focused upon the technique of ‘protein-film first demonstrated reversible, diffusion-controlled voltammetry voltammetry’, in which the protein molecules under in- of cytochrome c at a solid electrode without mediators and with vestigation are adsorbed on the electrode surface and no sign of denaturation. The all-important feature is application electrochemically ‘interrogated.’The experiments address a of a controlled electrochemical potential which is varied either minuscule sample with high sensitivity, and optimal control continuously or in steps or pulses: this activates specific redox over both potential and time dependence of reactions. centres, the reactions of which are measured simultaneously Factors governing the voltammetric response are outlined, through the current response, thereby procuring interdependent and particular emphasis is given to the ability to study thermodynamic and kinetic information from a single set of reactions that are coupled to and may ‘gate’ the primary experiments. The interactive nature of the experiments is electron exchange processes. Examples described include optimised by confining the sample under investigation to the proton-transfer and metal-binding reactions of iron-sulfur electrode surface, in a configuration which we refer to as clusters, coupling of electron transfer in peroxidases, quanti- ‘protein-film voltammetry’ and upon which we will focus our fying electron-transport pathways in multi-centred enzy- discussion.
    [Show full text]
  • A New Electrochemical Cell with a Uniformly Accessible Electrode To
    A new electrochemical cell with a uniformly accessible electrode to study fast catalytic reactions Mariam Fadel, Daurelle Jean-Vincent, Vincent Fourmond, Jérôme Vicente To cite this version: Mariam Fadel, Daurelle Jean-Vincent, Vincent Fourmond, Jérôme Vicente. A new electrochemical cell with a uniformly accessible electrode to study fast catalytic reactions. Physical Chemistry Chemical Physics, Royal Society of Chemistry, 2019, 21 (23), pp.12360-12371. 10.1039/c9cp01487j. hal- 02308533 HAL Id: hal-02308533 https://hal.archives-ouvertes.fr/hal-02308533 Submitted on 29 Jan 2020 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. A New Electrochemical Cell with uniformly Accessible Electrode to Study Fast Catalytic Reactions Mariam Fadel*1,2, Jean-Vincent Daurelle*1, Vincent Fourmond2, Jérôme Vicente1 1Laboratoire IUSTI (UMR AMU-CNRS 7343) Polytech Marseille, Dpt Mécanique Energétique (ME) Technopôle de Chateau Gombert 5 rue Enrico Fermi 13453 Marseille cedex 13 2Aix-Marseille Université, CNRS, BIP UMR 7281, 31 chemin J. Aiguier, F-13402 Marseille cedex 20, France *Email : [email protected] , [email protected] Abstract Electrochemical study of fast catalytic reactions is limited by mass transport using the conventional electrochemical cell of Rotating disk electrode (RDE) [4].
    [Show full text]
  • Chapter 2: the Ph-Dependent Redox Inactivation of Amicyanin As Studied by Rapid Protein- Film Voltammetry
    Chapter 2 Chapter 2: The pH-dependent redox inactivation of amicyanin as studied by rapid protein- film voltammetry Abstract The redox properties of the blue copper protein amicyanin have been studied with slow and rapid scan protein-film cyclic voltammetry. At slow scan rates, when the system is able to equilibrate with the electrode potential, the reduction potential of amicyanin depends on pH in a sigmoidal manner. The data are analysed by assuming that a single titrating group with a redox state dependent pKa is responsible for the pH-dependence of red ox the reduction potential, which gave pKa = 6.3, pKa £ 3.2 at 22 °C. Voltammetry at higher scan rates shows that the low-pH form of amicyanin is not oxidised directly. Instead, oxidation occurs only after conversion to the high-pH form. Simulations show that this conversion is achieved with a rate constant > 750 s-1 at 25 °C. In order to slow the coupled reaction down, the experiments were performed at 0 °C, at which a rate constant of 35 ± 20 s-1 was determined. In conjunction with evidence from NMR, the data are discussed within a scheme that incorporates protonation and dissociation of the copper coordinating histidine 96 in the reduced form. 27 Protein film voltammetry on amicyanin Introduction This paper deals with the dynamics of the pH-dependent conformational change that occurs in the active site of the small blue copper protein, amicyanin. Amicyanin functions as the obligatory electron acceptor of methylamine dehydrogenase (MADH) [Husain et al., 1985; Tobari et al., 1981; van Houwelingen et al., 1985; van Spanning et al., 1990], an enzyme that is essential when bacteria like Paracoccus versutus are grown on mimimal medium with methylamine as the sole source of carbon, nitrogen and energy [Dennison et al., 1998].
    [Show full text]