Inhibition studies of metalloproteins by means of electrochemistry and spectroscopy Anton Nikolaev To cite this version: Anton Nikolaev. Inhibition studies of metalloproteins by means of electrochemistry and spectroscopy. Other. Université de Strasbourg, 2018. English. NNT : 2018STRAF053. tel-02146028 HAL Id: tel-02146028 https://tel.archives-ouvertes.fr/tel-02146028 Submitted on 3 Jun 2019 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. UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES CHIMIQUE UMR7140, Chimie de la matière complexe THÈSE présentée par : Anton Nikolaev soutenue le : 29 octobre 2018 pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline/ Spécialité : Chimie Etudes d’inhibition de métalloprotéines par électrochimie et spectroscopie Inhibition studies of metalloproteins by means of electrochemistry and spectroscopy THÈSE dirigée par : Pr HELLWIG Petra, Université de Strasbourg, Strasbourg, France RAPPORTEURS : Pr MIOMANDRE Fabien, Ecole normale supérieure de Cachan, Paris, France Dr LOJOU Elisabeth, BIP CNRS AMU, Marseille, France _______________________________________________________________ AUTRES MEMBRES DU JURY : Dr KIEFFER Bruno, Université de Strasbourg, Strasbourg, France 1 Content Content ........................................................................................................................... 2 Acknowledgments .......................................................................................................... 6 Abbreviations and acronyms ......................................................................................... 7 Resumé en francais........................................................................................................ 9 Préface ............................................................................................................................................................... 9 Partie A. Cytochrome bd-oxydase ...................................................................................................................... 9 Partie B. La protéine mitoNEET ...................................................................................................................... 16 List of the communications ......................................................................................... 18 List of the publications ................................................................................................ 18 Preface .......................................................................................................................... 19 Part A. Cytochrome bd-oxidase ................................................................................... 20 Introduction ..................................................................................................................................................... 20 I. The place of complex IV in the respiratory chain ................................................................. 21 1. Bioenergetic membranes ................................................................................................................. 21 2. Components of respiratory chain .................................................................................................... 22 Eukaryotic respiratory chain ................................................................................................ 22 Cytochrome proteins and heme cofactors ............................................................................. 24 Prokaryotic respiratory chains ............................................................................................. 25 ROS ....................................................................................................................................... 25 Quinols .................................................................................................................................. 26 3. Cytochrome bd is a prokaryotic complex IV ................................................................................... 27 Classification of complex IV ................................................................................................. 28 Structure of complex IV......................................................................................................... 29 A-type oxidases ................................................................................................................. 29 B- and C-type oxidases, NORs .......................................................................................... 31 bd-oxidases ....................................................................................................................... 32 Catalytic cycle ....................................................................................................................... 35 aa3-oxidase ........................................................................................................................ 35 bd-oxidase ......................................................................................................................... 37 Function ................................................................................................................................ 38 II. Inhibitors: role in the protein characterisation .................................................................... 39 1. Kinetic parameters of enzyme catalysis .......................................................................................... 39 2. Influence of the inhibitors on enzyme kinetics Inhibitor classification .......................................... 41 3. Inhibition measurements ................................................................................................................. 43 III. bd-oxidase and inhibitors .................................................................................................. 45 IV. Development of electrochemical biosensor for the detection of inhibitors ....................... 49 1. Biosensors ....................................................................................................................................... 49 2 2. Protein film voltammetry ................................................................................................................ 50 Practical aspects ................................................................................................................... 50 Choice of the surface modification ................................................................................... 50 Gold electrode modified with GNPs covered with SAM ................................................... 54 Comparison of immobilised and solubilised complex IV ...................................................... 54 The aims of the project .................................................................................................................................... 56 Results .............................................................................................................................................................. 57 I. Optimisation of the cyt bd immobilisation. ........................................................................... 58 1. Influence of concentration .............................................................................................................. 58 2. Stability of the signal ...................................................................................................................... 59 Influence of the lipids ............................................................................................................ 60 Desorption or loss of the activity? ........................................................................................ 61 Mutual influence of the surface charge of the SAM and lipid type ....................................... 61 Influence of the thiol spacer length ....................................................................................... 63 Detergent variations ............................................................................................................. 64 3. Improvement of the preparation procedure .................................................................................... 65 GNPs deposition ................................................................................................................... 65 Thiol incubation .................................................................................................................... 67 The protein incubation at the surface ................................................................................... 69 4. Control experiments with HQNO .................................................................................................... 69 5. Conclusions..................................................................................................................................... 71 II. The compound examination. ................................................................................................