NANOSCALE STUDIES OF METAL REDUCTION BY MICROBIAL BIOCATALYSTS USING IN VITRO BIOMIMETIC PLATFORMS By Bhushan Prabhakar Awate A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemical Engineering—Doctor of Philosophy 2014 ABSTRACT NANOSCALE STUDIES OF METAL REDUCTION BY MICROBIAL BIOCATALYSTS USING IN VITRO BIOMIMETIC PLATFORMS By Bhushan Prabhakar Awate Metal-reducing bacteria like Geobacter sulfurreducens use cytochrome proteins to reductively precipitate water-soluble uranium salts. However, the mechanism by which the cytochromes achieve multistep electron transfer to extracellular metals is not yet understood. Previous studies of cytochromes' role in electron transfer have involved a genetic approach, in which specific cytochromes are either deleted or overexpressed. However, results of these genetic studies are difficult to interpret, because mutation of one gene can cause multiple phenotypic changes, resulting in complex alterations of the cell's electron-transfer machinery. These limitations can be bypassed using a biomimetic approach, in which Geobacter cytochromes are assembled into nanostructured interfaces that mimic the cell envelope and electron-carrier machinery. In this study, we heterologously expressed some of Geobacter’s most abundant and conserved cytochromes in Escherichia coli. We then used these cytochromes to fabricate nanostructured biomimetic interfaces that mimicked Geobacter’s double-membrane cell envelope. A self-assembled monolayer of alkanethiols on a gold electrode mimicked the inner membrane; an aqueous layer containing PpcA (a periplasmic cytochrome) mimicked the periplasmic space; and a synthetic bilayer lipid membrane containing OmcB (an outer membrane cytochrome) mimicked the outer membrane. Cytochrome-mediated electron transfer from the gold electrode to soluble metal salts was characterized using cyclic voltammetry. The PpcA was found to transfer electrons to U(VI) more rapidly than to other soluble electron acceptors, consistent with the observation that U(VI) is reductively precipitated in Geobacter’s periplasm. Spectroelectrochemical characterization of PpcA and OmcB demonstrated for the first time electron transfer between these two proteins, suggesting that they may be redox partners in Geobacter’s electron transport chain. Fabrication of electrochemically active nanostructured bioelectronic interfaces that mimic Geobacter’s double-layered cell envelope establishes a new experimental platform with which to characterize Geobacter’s electron transfer machinery. Addition of more Geobacter components will make the interface more realistic and enable hypotheses about electron-transfer mechanisms to be systematically tested. An improved understanding of Geobacter’s ability to reduce metals may lead to new technologies for in situ reductive immobilization of uranium and other toxic metals. ACKNOWLEDGEMENTS I would like to thank my advisor Dr. R. Mark Worden for all the help and guidance he provided for this research. And I would also like to thank him for being patient and supportive during the challenging times I faced. I wish to convey thanks to Dr. Gemma Reguera for providing me the opportunity to work with this extremely interesting multidisciplinary project. I wish to thank Dr. Scott Calabrese - Barton for his time and help he provided me as a committee member and also as a graduate coordinator. I would also like to thanks Dr. Ilsoon Lee for his helpful suggestions and for providing expertise in nanotechnology area. I would also like to give special thanks to Dr. Denise Proshlyakov and his graduate student Chris John for helping and guidance me regarding the spectrophotometric techniques. My work here has been possible because of the help form fellow graduate students and post docs in my and my collaborator's labs. therefore, I would like to thank Lee Alexander, Chloe, Ying, Becky, Mike, Blaire and Dr. Jihwan. I would also like to convey my gratitude to Dr. Alicia Pastor for help regarding TEM, and Dr. Yuri Londer for providing me plasmid for protein expression. And finally last but not the least, I would like to thank my family for being extremely supportive. iv TABLE OF CONTENTS LIST OF TABLES ..................................................................................................................... viii LIST OF FIGURES ..................................................................................................................... ix KEY TO ABBREVIATIONS..................................................................................................... xii 1.0 INTRODUCTION................................................................................................................... 1 1.1 Motivation ................................................................................................................. 1 1.2 Background ............................................................................................................... 2 1.3 Problem description .................................................................................................. 4 1.3.1 Heterologous expression of cytochrome in E. coli ............................................ 6 1.3.2 Electrochemical measurements .......................................................................... 8 1.3.3 Spectro-electrochemical measurements ............................................................. 9 1.4 Dissertation Outline ................................................................................................ 10 2.0 EXPRESSION, PURIFICATION, IMMOBILIZATION AND ELECTROCHEMISTRY OF RECOMBINANT PPCA ........................................................ 11 2.1 Introduction............................................................................................................. 11 2.1.1. Immobilization of PpcA on biomimetic interface........................................... 11 2.1.2 Electrochemical characterization ..................................................................... 12 2.2 Materials and methods ............................................................................................ 13 2.2.1 Production of PpcA .......................................................................................... 13 2.2.2. Purification of PpcA ....................................................................................... 14 2.2.3 Immobilization of PpcA on biomimetic interfaces .......................................... 15 2.2.4 Electrochemical measurements ........................................................................ 15 2.2.5 Ellipsometric measurements ............................................................................ 16 2.3 Results and discussion ............................................................................................ 16 2.3.1 Production and purification of PpcA ............................................................... 16 2.3.2 Electrochemistry of PpcA in solution .............................................................. 17 2.3.3 Immobilization of PpcA and its electrochemistry ........................................... 18 3.0 ELECTROCHEMICAL CHARACTERIZATION OF GEOBACTER SULFURREDUCENS PPCA C-CYTOCHROME USING BIOMIMETIC INTERFACES REVEALS GREATER REDUCTION RATES OF URANIUM THAN IRON .................... 22 3.1 Abstract ................................................................................................................... 22 3.2 Introduction............................................................................................................. 22 3.3 Materials and Methods............................................................................................ 25 3.3.1 Recombinant PpcA expression and purification. ............................................. 25 3.3.2 Biomimetic interfaces with PpcA. ................................................................... 25 3.3.3 Electrochemical measurements. ....................................................................... 25 3.4 Results and Discussion ........................................................................................... 29 3.4.1 Catalytic activity of PpcA with iron and uranium salts. .................................. 29 3.4.2 PpcA exhibits high second order rate constants (ko’) for the reduction of U. .. 31 v 3.4.3 Specific interactions between U and PpcA promote reduction rates. .............. 33 3.5 Implications ............................................................................................................ 36 4.0 HETEROLOGOUS EXPRESSION OF OMCB IN E. COLI AND ITS LOCALIZATION TO BACTERIAL MEMBRANE .............................................................. 39 4.1 Abstract ................................................................................................................... 39 4.2 Introduction............................................................................................................. 39 4.3 Methods .................................................................................................................. 41 4.3.1 Sequence data................................................................................................... 41 4.3.2 Bacterial strains and plasmids .......................................................................... 42 4.3.3 Subcellular fractionation .................................................................................. 44 4.3.4 Mass spectroscopy ........................................................................................... 46 4.3.5 Biochemical characterization with UV visible spectroscopy .......................... 47 4.5 Results and discussion ............................................................................................ 47 5.0 SPECTROELECTROCHEMICAL STUDIES OF GEOBACTER REDOX PROTEINS EXPRESSED IN E. COLI .......................................................................................................... 54 5.1 Introduction............................................................................................................. 54 5.2 Materials and methods ............................................................................................ 56 5.2.1 Thin-layer spectroelectrochemical system ....................................................... 56 5.2.2 PpcA and OmcB cytochrome c ........................................................................ 57 5.2.3 Electrochemical and Spectroscopic measurements.......................................... 57 5.2.4 Preparation of working electrode ..................................................................... 58 5.3 Results and Discussion ........................................................................................... 59 5.3.1 Immobilization of PpcA on optically transparent gold electrodes................... 59 5.3.2 Spectroelectrochemical characterization of PpcA in solution ......................... 60 5.3.3 Spectroelectrochemical characterization of OmcB in OTTLE ........................ 62 5.3.4 Spectroelectrochemical characterization of PpcA -OmcB interactions ........... 63 6.0 ENGINEERING A BIOMIMETIC REDOX ACTIVE CELL ENVELOPE WITH HETEROLOGOUSLY EXPRESSED GEOBACTER CYTOCHROMES ............................ 66 6.1 Introduction............................................................................................................. 66 6.2 Materials and methods ............................................................................................ 68 6.2.1 Preparation of protein samples......................................................................... 68 6.2.2 Preparation of DOPC liposomes ...................................................................... 69 6.2.3 Fabrication of compartmentalized bilipid membrane on gold electrode ......... 69 6.2.4 Analytical techniques ....................................................................................... 70 6.3 Results and discussion ............................................................................................ 72 6.3.1 Characterization of DOPC liposomes and microsomes ................................... 72 6.3.2 Characterization of BRACE using QCM ......................................................... 72 6.3.3 Electrochemical characterization of BRACE .................................................. 73 6.4 Implications and future work .................................................................................. 76 7.0 CONCLUSIONS ................................................................................................................... 80 vi APPENDICES ............................................................................................................................. 83 APPENDIX A: OMCB EXPRESSION AND PURIFICATION ................................ 84 APPENDIX B: FIGURES AND TABLES .................................................................... 88 REFERENCES.......................................................................................................................... 135 vii LIST OF TABLES Table 3.1: ko’ values for different metal acceptors obtained by cyclic voltammetry and rotating disk electrode method. ................................................................................................................ 105 Table 4.1: List of different plasmids used for OmcB expression in E. coli. ............................... 112 Table 6.1: Reduction potentials and free energies of redox compounds .................................... 129 Table A .1: List of some of the detergents tried to solubilize OmcB ......................................... 130 viii LIST OF FIGURES Figure 1.1: The pathways of energy metabolism conserved across different species of Geobacter. ....................................................................................................................................................... 89 Figure 1.2: The model for dissimilatory metal reduction by Geobacter. Energy is conserved by proton pumping across inner membrane. ...................................................................................... 90 Figure 1.3: Transfer of electron across bacteria along the potential gradient. .............................. 91 Figure 2.1: A. Chromatogram of PpcA capture step from the periplasmic fraction using cation exchange resin............................................................................................................................... 92 Figure 2.2: Purification of recombinant PpcA using cation exchange chromatography with a NaCl gradient ................................................................................................................................ 93 Figure 2.3: Dithionite-reduced minus oxidized spectra of recombinant PpcA protein ................ 94 Figure 2.4: CV of PpcA in solution at SAM covered gold electrode ........................................... 95 Figure 2.5: Cyclic voltammograms of PpcA in solution on a SAM coated gold electrode at increasing scan rates ..................................................................................................................... 96 Figure 2.6: A. Cyclic voltammograms of PpcA immobilized on a SAM-functionalized gold electrode at increasing scan rates .................................................................................................. 97 Figure 2.7: Trumpet plot obtained from the change in cathodic and anodic peak potential at various scan rates in CV experiments ........................................................................................... 98 Figure 3.1: Conceptual design of the biomimetic interface .......................................................... 99 Figure 3.2: Cyclic voltammograms of PpcA interfaces in the presence (black) or absence (grey) of 200 µM Fe-NTA (A) or U acetate (B).................................................................................... 100 Figure 3.3: Calculation of second order rate constants ............................................................... 101 Figure 3.4: Rate constant values ................................................................................................ 102 Figure 3.5: RDE voltammogram of PpcA immobilized on a SAM-functionalized gold electrode in the presence of 400 µM Fe-NTA at different rotation speeds ................................................ 103 Figure 3.6: (A) Linear correspondence between cathodic currents (reverse of steady state current values or iss) of PpcA immobilized on a SAM-functionalized rotating disc gold electrode and the -1/2 rotations speed (ω ) for different concentrations of Fe-NTA ................................................. 104 Figure 4.1: OmpA-OmcB Amino acid sequence ........................................................................ 106 ix Figure 4.2: SDS PAGE profile of OmcB expression and cell fractionation ............................... 107 Figure 4.3: Ultracentrifugation with sucrose density gradient separates OmcB membrane fractions based on buoyant density ............................................................................................. 108 Figure 4.4: Whole cell spectroscopy of E. coli BL21 cells expressing OmcB ........................... 109 Figure 4.5: OM fragments containing OmcB reduced different metal samples ......................... 110 Figure 4.6: Cysteine mutation at 1 cysteine to alanine affected redox property of the OmcB ... 111 Figure 5.1: A. Schematic diagram of an OTTLE cell. ................................................................ 113 Figure 5.2: Square wave voltammogram of PpcA adsorbed on SAM coated transparent gold electrode ...................................................................................................................................... 114 Figure 5.3: A. Cyclic voltammograms of PpcA in solution on a SAM coated gold electrode at OTTLE with increasing scan rates.............................................................................................. 115 Figure 5.4: UV-vis spectroscopic response of PpcA at the OTTLE ........................................... 116 Figure 5.5: A. Chronoamperometry of PpcA at OTTLE ............................................................ 117 Figure 5.6: Subtraction spectra of OmcB with mediator (AQDS 50 µM) at OTTLE during the CV run at 1 mV/s ........................................................................................................................ 118 Figure 5.7: Spectroelectrochemical characterization of OmcB heme potentials using redox mediators ..................................................................................................................................... 119 Figure 5.8: AQDS mediated reduction of OmcB membrane vesicles at OTTLE monitored by spectroscopic technique .............................................................................................................. 120 Figure 5.9: Cyclic voltammogram of PpcA with and without OmcB membrane vesicles at 50 mV/s ............................................................................................................................................ 121 Figure 6.1: A. TEM images of DOPC liposomes prepared by sonication .................................. 123 Figure 6.2: A. The phase angle and log z data plotted against log frequency for the bilayer made from proteoliposomes ................................................................................................................. 124 Figure 6.3 A. CV of BRACE formed with PpcA and DOPC bilayer ......................................... 125 -heme Figure 6.4 A. CV of BRACE formed with PpcA and OmcB proteoliposomes. ................ 126 Figure 6.6: Schematic representation of Geobacter cell envelope (A) and Biomimetic redox active cell envelope (B). ............................................................................................................. 128 Figure A.1:A, B: TEM images of E. coli BL 21 cells expressing OmcB protein...................... 132 x Figure A.2: A. Agarose gel electrophoresis of pMAL plasmids digested with EcoRI and NdeI restriction enzymes ..................................................................................................................... 133 Figure A.3: A. Gel filtration chromatography of urea denatured OmcB with superdex 200TM resin ............................................................................................................................................. 134 xi KEY TO ABBREVIATIONS Chemicals ACN Acetonitrile Ag/AgCl Silver/silver chloride ATP Adenosine triphosphate CaCl2 Calcium chloride DMR Dissimilatory metal reducing microorganisms DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine DTT Dithiothreitol EDC 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide EDTA Ehtylenediamine tetraacetic acid HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid IPTG Isopropyl-β-D-thiogalactopyranoside KCl Potassium chloride LB Luria Bertani MgCl2 Magnesium chloride NaCl Sodium Chloride NHS N-hydroxysuccinimide NTA Nitrilotriacetic acid TFA Trifluoroacetic acid xii UA Uranyl acetate Symbols A Electrode area CDL Double layer capacitance CF Faradaic capacitance CM Membrane capacitance CPE Constant phase element D Diffusion coefficient F Faradays constant iP Peak faradaic current kDA Kilodalton Q Charge transferred during oxidation/reduction RCT Charge transfer resistance RM Membrane resistance RS Solution resistance t Time τ Surface coverage T Temperature v Scan rate xiii V Applied potential Y Admittance Z Impedance ZI Imaginary part of impedance ZR Real part of impedance θM Phase delay ω Frequency Terminologies BLM Bilayer lipid membrane BRACE Biomimetic redox active cell envelope DET Direct electron transfer ET Electron transfer SAM Self assembled monolayer sBLM Supported BLM tBLM Tethered BLM Instrumentation CA Chronoamperometry CV Cyclic voltammetry DLS Dynamic light scattering EIS Electrochemical impedance spectroscopy xiv MS Mass spectrometry QCM Quartz crystal microbalance TEM Transmission electron microscopy UV-vis Ultraviolet- visible xv 1.0 INTRODUCTION 1.1 Motivation Anthropogenic use of uranium has lead to the contamination of vast areas of subsurface waters and sediments, resulting in an environmental hazard. (1) More than 100 uraniumcontaminated sites has been identified by U.S. Department of Energy (DOE) as a target for remediation. (2) Ex situ pump and treat decontamination methods involve sorption, volatilization, and biotic or abiotic treatments along with groundwater flushing. Disadvantage of these techniques is that they cannot be applied to the larger area with low localized concentration of contaminants and their high cost. (3) In situ bioremediation is proposed to be the most cost effective technique to clean up subsurface and groundwater contamination. (1) Bioelectronic reduction of uranium from soluble U (VI) form to insoluble (IV) form decreases its mobility and can limit the spread of uranium contamination through the groundwater. (4) Lab-scale as well as at pilot-scale field studies found that microbes from the bacterial family Geobacteraceae were instrumental in this bioremediation process. (5-10) Stimulating the activity of metal-reducing bacteria such as Geobacter spp. in situ shows promise for the immobilization of uranium, which is concomitantly reduced to its less soluble form, U(IV). (11) The reduction of U(VI) by Geobacter is concomitant to the reduction of Fe(III) oxides, a process that relies on the extracellular transfer of metabolically-generated electrons across electron carriers of the cell envelope. However, Geobacter’s mechanism of biological metal reduction is still not understood at the molecular level. The objective of our project is to elucidate the electron transfer (ET) mechanism of the Geobacter using a nanostructured biomimetic interface. 1 1.2 Background Dissimilatory metal reducing microorganisms (DMR) like Geobacter obtain energy by oxidizing organic compounds inside the cell and then transferring the electrons to the electron acceptors (mostly metal oxides) located outside the cell. Genome sequence analysis of different Geobacter species have identified some of conserved respiratory pathways (Fig. 1.1). (12) Electrons obtained from oxidation of organic carbon are used to reduce electron carriers like + NAD , which transfer the electrons to the menaquinone pool. Then these electrons travel from the inner membrane of bacteria, across the periplasm with the help of number of periplasmic cytochrome proteins. It was postulated that heme network (cytochrome proteins) exists in this bacterium to either shuttle the electrons to the extracellular electron acceptors or to serve as an electron-storage depot in the absence of them. (13) A periplasmic cytochrome PpcA which is conserved across different families of Geobacter is thought to be most important of them. (14, 15) The electrons are passed by the periplasmic cytochromes to the outer membrane cytochromes, and finally to the electron acceptors outside the cell. Although the overall framework of energy metabolism inside the cell remains same in most of the DMRs, ET to extracellular electron acceptors can occur through a variety of mechanisms, including (1) indirect ET by solubilization of iron with organic chelators, (2) indirect transfer using electron shuttles (3) direct ET from the outer membrane c-cytochromes, and (4) direct ET with microbial protein nanowires - pili. The first mechanism has been claimed to be the mode of ET in Shewanella putrefaciens (16, 17), still this mechanism is yet to be accepted without reservations as a mode of ET in different Shewanella species. In Shewanella, the second mechanism is thought to be more predominant as Shewanella secrete soluble electron shuttles like riboflavin and flavin mononucleotides. (18, 19) However Geobacter species do not produce small shuttling 2 molecules, and electron shuttle with cytochrome secretion may not be metabolically economical for the bacteria. A lot of emphasis has been given to the third model due to the large number of c- cytochromes present in the Geobacter and Shewanella species, which are capable of reducing iron in vitro. (20-23) G. sulfurreducens has been shown to transfer electrons to an electrode via its outermost membrane cytochromes. (24) Various studies have well documented the important roles of different outer membrane cytochromes like OmcZ (25, 26), OmcB (27), OmcS, and OmcT (28) in Geobacter ET. However, very little homology exists among different species of Geobacter and related Pelobacter species regarding the content of cytochrome related genome, yet they all are capable of growing with Fe (III) oxide, and are present in uranium contaminated environments. (29) While these variances create uncertainties about the exact role of outer membrane cytochromes, a highly conserved pili protein emerge as a likely candidate for final electron transfer in these organisms. Pili are the hair like appendages present on the surface of many bacteria, and are involved in DNA transfer, motility and surface attachment. G. sulfurreducens specifically produces pili during the growth on insoluble metal salts. (30) When pilA gene was deleted, G. sulfurreducens could not produce pili and failed to reduce insoluble electron acceptors such Fe(III) oxides. However these mutants could still use soluble electron acceptors like Fe(III) citrate and fumarate. These results point out that the pili might have a more direct role in electron transfer to Fe(III) oxides. (30) Geobacter pili were analyzed with a conductive probe atomic force microscope (AFM) and found to be conductive. This suggests a crucial role of pili in Geobacter for Fe(III) oxide reduction, as pili can serve as a nanowires to conduct electron transfer between the cell surface of the Fe(III) oxides. The exact nature of conduction of electrons along the length of pili is still under investigation. It was hypothesized that OmcS is 3 localized along the length of pili and the OmcS is responsible for the ET from pili to Fe(III) oxide. (31) However this interpretation is questionable, because a recent publication has shown that pili are primarily responsible for the extracellular reduction and localization of the uranium. (32) The Geobacter strains with mutation designed to produce higher amount of pili, reduced more uranium than native wild type pili, but OmcS content of both the strains were same, suggesting the pili mediated ET is not directly dependent on OmcS expression. (32) Studies carried out to measure the conductivity of these microbial nanowires has verified the conductive nature of pili. (33) A recent study showed that Geobacter strain with deletion of multiple outer membrane cytochrome decrease the rate of extracellular U reduction. (34) It is possible that one of the deleted cytochrome may be electron donor to pili. Still the mechanism by which pili receive the electrons remains unidentified, and is under investigation. Figure 1.2 represents a schematic description of our understanding of the Geobacter ET process based on current literature review. (22, 35, 36) 1.3 Problem description The theoretical energy gained (∆G) for bacteria is directly related to the potential difference between electron donor and acceptor: ∆𝐺 = −𝑛 𝐹𝐸𝑒𝑚𝑓 where n is the number of electron exchanged, F is Faraday's constant and Eemf is the potential difference. Bacteria will try to maximize the energy gain by selecting the electron acceptor with higher potential and electron donor with lower potential. Therefore it is crucial to gain knowledge about thermodynamic properties of this electron transport carrier proteins to understand their significance in bacterial ET. A putative picture of energy coupling with ET 4 process is shown in Figure 1.3. Biological approaches to identify these electron carriers are limited by pleiotropic effects resulting from alterations in the native uranium reductase machinery. For example, deletion of OmcF gene also led to reduced expression of OmcB protein, and resultant cells showed impaired growth on Fe (III) citrate (37) Absence of OmcG and OmcH, did not affect the transcript levels of OmcB; however, expression of OmcB was affected negatively in those strains. (38) Deletion of OmcB led to reduced growth of the cells, but cells eventually recovered with increased expression of other outer membrane cytochromes like OmcS and OmcT. (39) These pleiotropic constraints can be bypassed through the use of in vitro platform that mimic the Geobacter cell membrane electron carrier machinery. The objective of this research was to construct biomimetic redox active cell envelope (BRACE) containing Geobacter’s most abundant and conserved electron carriers to mimic Geobacter cell membrane electron carrier machinery. We identified periplasmic cytochrome protein PpcA and an outer membrane cytochrome OmcB as an integral components of designed biomimetic interface. These proteins were expressed heterologously in E. coli host in order to achieve high yields of proteins necessary for the study, and to isolate the target cytochromes from bacteria without any interference from other redox proteins. The BRACE interfaces were assembled on a gold or carbon electrode (cathode) that act as a source of electrons. An example BRACE interface consist of (1) a self-assembled monolayer of lipids that simulate the inner membrane of Geobacter; (2) a layer containing periplasmic cytochrome PpcA, which act as an intermediate electron carrier to either outer membrane cytochrome and/or pili; (3) a lipid layer, which act as an outer membrane and will insulate the PpcA layer from the solution, thus creating an artificial periplasmic space; (4) an outer membrane cytochrome OmcB embedded in the lipid 5 layer; (5) a self assembled monolayer of lipids, with immobilized PpcA deposited on optically transparent electrode. Till date a very few studies are available about the kinetics of the reduction reaction of metal species and DRMs, (40-42) and even fewer studies are available about the kinetics of specific cytochromes from Shewanella and metals, (40, 43, 44) but none of the specific cytochromes from Geobacter has been analyzed for their kinetics of metal reduction. Our present research aims to bridge the knowledge gap about the putative role of these individual cytochrome in Geobacter ET. Furthermore our aim has been to provide a versatile biomimetic interface in the form of BRACE, which can be used to study different proteins, and which can be adapted to diverse application such as biosensor development. These novel biomimetic interfaces will provide the platform necessary to investigate the flow of electrons from Geobacter membrane to the electrons acceptors. Information gained from these studies will enhance understanding of Geobacter ET and may guide efforts to engineer uranium bioremediation strategies. 1.3.1 Heterologous expression of cytochrome in E. coli Heterologous expression of cytochrome in E. coli is highly desirable for the possibility of mass production and purification; but it can be very challenging because of the posttranslational modification required for the activity of these proteins. Therefore till date very few recombinant cytochromes have been expressed in E. coli. To best of our knowledge Shewanella cytochromes: MtrA (45), CymA (46), and OmcA (47) and Geobacter cytochrome PpcA (48) are the only cytochrome that has been successfully expressed in E. coli. Careful understanding of the cytochrome c biogenesis is required to design its heterologous expression strategy. 6 Cytochrome c proteins are hemoproteins in which iron-protoporphyrin IX (heme b) groups are covalently bound to the polypeptide chain. Cytochrome c proteins have at least one heme binding motif (C1XXC2H), where the cysteines forms two thioether bonds with vinyls at position 2 and 4 of the tetrapyrrole rings of heme b. (49) The histidine residue serves as one of the axial ligand to the heme-iron. Methionine or another histidine residue located farther away in amino acid sequence forms the other ligand. The stereo-selectivity of heme attachment to the cytochrome c polypeptide chain is same in all known examples of cytochrome c.(50) Heterologous expression of cytochrome c is challenging because none of the cytochrome c are cytoplasmic proteins. They are either expressed in the intermediates space of mitochondria or in the periplasmic space or are membrane associated via transmembrane helix or a lipid anchor. (51) For biogenesis of a functional cytochrome c, both apoprotein and heme moieties must transloacte through inner membrane. Then the apoprotein and heme must be brought together by series of enzymes and chaperon proteins to form a thioether bond. Cytochrome c biogenesis processes are still being discovered, but based on current knowledge about the proteins involved in the apocytochrome assembly and chaperoning, heme handling and thioether bond formation, they are divided into three classes: system I, II and III. (52) Many gram negative bacteria including E. coli use system I (also called the Ccm system) for cytochrome c assembly. The Ccm system employs up to ten different membrane bound components (CcmABCDEFGHI and DsbD/CcdA) whose operation can be divided into three different modules. (53) Module 1 consists of Ccm proteins A to E and are involved in the translocation and relaying of heme b. The exact mechanism of the heme translocation across the membrane is still unclear and not entirely dependent on presence of Ccm proteins (54) but Ccm proteins are essential for assembly of c-type cytochromes where heme is covalently bound. 7 Module 2 is involved in apocytochrome c thioreduction (to make it ligand competent) and chaperoning process. It is important that after translocation across the membrane the cysteine moiety of apocytochrome c destined to bind heme must be in reduced state in the oxidative environment of periplasmic space. This is proposed to be achieved by first oxidizing the cysteines with DsbA-DsbB pathway to form disulfide bond making it less susceptible to degradation. Then the cysteine is reduced by CcmG/CcmA pathway. Cysteines of CcmH are also involved although not directly in the reduction of cysteine of apocytochrome c. Module 3 consisting of CcmH, CcmF and CcmI is involved in stereoselctive ligation of apocytochrome c to heme b resulting in holocytochrome c. In E. coli the Ccm protein are located in the inner membrane and on the periplasmic side of inner membrane and are active only in anaerobic conditions. (36) Cloning of the gene cluster ccmABCDEFGH into pACYC184 plasmid enables its constitutive co expression under aerobic conditions. (55) In this study, pEC 86, a derivative of the pACYC184 plasmid was co expressed with cytochrome c plasmid in E. coli to achieve mature cytochrome c expression. 1.3.2 Electrochemical measurements Electrochemical techniques offer a powerful and convenient way of analyzing the redox proteins of interest in controlled environment. Some of the cytochromes are capable of direct electron transfer (DET) from the electrode without any additional redox mediators. We have taken advantage of this capability to study the kinetic and thermodynamic properties of these cytochrome on biomimetic interface using a variety of techniques like cyclic voltammetry, chronoamperometry, square wave voltammetry, and electrochemical impedance analysis. 8 1.3.3 Spectro-electrochemical measurements Spectroelectrochemical techniques combines electrochemistry and spectroscopic measurements, and offers advantage offering capability to assess changes in specific molecules in the system as a function of electric potential. We built working electrode form a transparent o slide coated with gold (~100 A thickness) and covered with self assembled monolayer (SAM) of thiolipids. Formation of SAM on the gold electrodes shielded the protein solution from degradation at the electrode surface, and furthermore provided us with the capability of controlling the access of species in solution towards working electrode. Redox activity of Geobacter cytochromes was measured electrochemically by technique like cyclic voltammetry and chronoamperometry, and multipotential analysis where changes in current at the working electrode are monitored as a function of applied potential. High transparency of the working electrode allowed us to measure reduction of cytochromes spectrophotometrically by presence of three characteristic absorption bands. Despite its enormous importance in bioremediation as well as its potential application in MFCs, the ET mechanisms in DRMs has not been established yet. We have identified the unmet need about the tools required to study the Geobacter ET in vitro. The research presented in this dissertation focuses on building biomimetic systems that will help bridge the knowledge gap in this field. We have recognized the challenges associated establishing the theory about the ET mechanism, and have made a conscious effort to build a novel BRACE system that will facilitate the understanding about these complex processes in a more controlled environment. 9 1.4 Dissertation Outline Chapter 2 of this dissertation describes a method to heterologously express and purify periplasmic cytochrome form Geobacter: PpcA in E. coli. Initial electrochemical characterization of the protein and method to immobilize it on electrode are presented. Kinetic of electron transfer reaction between protein and electrode are calculated. Chapter 3 describes the calculation of electron transfer kinetics between immobilized protein and soluble metal electron acceptors determined by cyclic voltammetry and rotating disk electrode technique. Chapter 4 describes our efforts to express Geobacter outer membrane protein OmcB in E. coli, confirmation of its identity and its localization to outer membrane of the E. coli. Chapter 5 describes development of spectroelectrochemical interface to study the redox active proteins on a thin layer electrode. Chapter 6 describes the formation of a BRACE interface with both PpcA and OmcB and its electrochemical characterization. Chapter 7 summarizes conclusions of this studies. 10 2.0 EXPRESSION, PURIFICATION, IMMOBILIZATION AND ELECTROCHEMISTRY OF RECOMBINANT PPCA 2.1 Introduction One hallmark feature of Geobacter species as revealed by examination of genome sequence of Geobacter sulfurreducens is the presence of more than 70 c-type cytochromes. (57) It has been hypothesized that c-cytochromes acts as intermediate electron carriers from cytoplasm to outer membrane and then pili proteins complete the circuitry between cells and electron acceptors. A family of periplasmic triheme cytochromes was identified, (58) which was thought to be responsible for the periplasmic reduction of iron (59) and possibly as an intermediate in the transfer of electrons to the electron acceptor outside the cells. (60) PpcA is one of the most abundant of these periplasmic cytochromes and gene knockdown studies have shown its importance in Fe (III) and U (VI) reduction. (15) We have chosen PpcA as a protein of interest in our studies because it is one of the most conserved periplasmic cytochromes across different Geobacter species. (14) PpcA is a small protein of molecular size about 9.6 kDa, made up of 71 amino acids, and a very high proportion of lysine residues. (58) This protein has been studied by X-ray diffraction, (58) and nuclear magnetic resonance (NMR) to elucidate its structure and redox thermodynamics in great detail. (61-63) (64) The study of 15 N labeled PpcA with NMR spectroscopy was used to elucidate the structure of the protein, its backbone dynamics and the conformational changes based on the pH of the solution. (65) However, identity of the redox partner of PpcA in Geobacter ET is unknown. 2.1.1. Immobilization of PpcA on biomimetic interface The studies mentioned in the previous section were done with the protein in solution. However, the biological compartments are much more viscous than the buffer solutions in which 11 these studies were done. For example, PpcA is located in the periplasm of bacteria, which environment resembles more like a thick gel (66), and the width of this space is about 10-25 nm. (66, 67) The diffusion processes inside the cell are slower for the biomolecules and even much slower for the proteins. (68, 69) Therefore, to better mimic the biological conditions inside the cells, we developed a protein immobilization strategy. The literature reviews showed large number of strategies available to immobilize cytochrome c on electrode; for example with Nacetyl cysteine, (70) cysteamine-modified gold with EDC coupling, (71) polypyrrole matrix, (72) functionalized vertically aligned carbon nanofiber scaffold, (73) beta-cyclodextrin-modified gold nanoparticles, (74) and even bare gold (75) to name just a few. We chose a self assembled monolayer (SAM) of alkanethiols on gold electrode as our substrate for immobilization. Gold oxides are formed on the surface of the electrode by the method employed to clean the surface (such as oxygen plasma cleaning), which are reduced by thiol terminated alkanes which self assemble on the gold electrode. (76, 77) Immobilization of cytochrome on this SAM is preferable because based on the functional group (e.g. carboxyl, hydroxy), and the chain length of carbon the chemistry of SAM can be fine-tuned to obtain optimum immobilization. (78-83) 2.1.2 Electrochemical characterization Electrochemical characterization of interface was done by technique called cyclic voltammetry (CV). In CV technique, the voltage is swept between two potential values at a fixed scan rate. (84) In the forward scan, potential is swept from voltage V1 to voltage V2. Once the voltage reaches V2 the scan is reversed and the voltage is swept back to V1. The current is plotted as a function of applied voltage in a cyclic voltammogram. The shape of the voltammogram depends on the quality of electrode surface and the mass transfer effects. The redox couple present at the electrode surface get reduced or oxidized at the standard redox 12 potential value for the corresponding redox reaction. CV measurements can be used to determine the surface coverage of the immobilized redox species, diffusion coefficient of the redox species in the solution, and to gain insights into the mechanism of the reaction (such as multistep reactions). 2.2 Materials and methods 2.2.1 Production of PpcA PpcA was heterologously expressed in Escherichia coli strain BL21 (DE3), as previously reported. (48) E. coli cells were co-transformed with plasmids pEC 86 and pCK 32. Plasmid pEC 86 contains the ccm genes that enable heme incorporation and maturation of PpcA in E. coli. (85) Plasmid pCK 32 plasmid is a derivative of plasmid pkIVlen004 with the ppcA fused to the signal peptide of the E. coli OmpA leader sequence for periplasmic expression of PpcA. Cells were grown in 2X YT liquid or solidified media containing 100 µg/ml ampicillin and 34 µg/ml chloramphenicol. The cultures used for purification of PpcA were initiated by growing a single colony of freshly co-transformed E. coli cells in 50 ml of 2x YT liquid medium supplemented o with antibiotics. After overnight incubation at 30 C with 250 rpm gyratory agitation, 10 ml of the culture were transferred into a 1 L of fresh medium and incubated under the same conditions to an optical density at 600 nm of approximately 1. PpcA expression was then induced with 100 µM of isopropyl β-D-1-thiogalactopyranoside (IPTG) during overnight incubation. Expression of PpcA was monitored by taking a sample of the inoculated fermentation media during the growth centrifugation at 5000X G to get a cell pellet. The pellet was digested by O.D. normalized value of Laemmle buffer with SDS, and running a SDS PAGE. Cells were harvested by centrifugation o (4000 g, 4 C, 15 min). 13 2.2.2. Purification of PpcA Periplasmic protein fractions were separated by osmotic shock using lysis buffer containing 100 mM Tris-HCl, pH 8, 0.5 mM EDTA, 20% sucrose and 0.5 mg/ml lysozyme. Cells were ground gently using a rubber scraper and incubated at room temperature for 15 min. After addition of ice-cold water, the cells were incubated on ice for 15 min with gentle agitation. o The periplasmic fraction was recovered in supernatant fluid after centrifugation (12000 g, 4 C, 20 min) and filter-sterilized through 0.2 µm syringe filters. PpcA was separated from other periplasmic proteins in the supernatant fluid by cation exchange chromatography using a SP Sepharose ® fast flow column (GE health care) equilibrated with 20 mM Tris-HCl pH 8.5 and eluted with a step salt gradient with 20 mM Tris-HCl, pH 8.5, 1 M NaCl at flow rate of 2 ml/min. Excess salt was removed from the sample using gel filtration column. Final purification was done using the same cation exchange column but eluting the protein with a 0-300 mM NaCl salt gradient in 10 mM Tris HCl, pH 8.5. Five ml fractions eluted from the column were collected, and their protein and heme content was monitored spectrophotometrically as UV absorbance at 210 nm (peptide bonds) and 408nm (heme). Those fractions having ratio of A408/A210 close to 1 were pooled together, and their protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 4-15 % Biorad Tris Glycine precast gel and Coomassie blue staining. The redox activity of the purified PpcA protein was confirmed by dithionite-reduced minus air-oxidized difference spectra (420-700 nm), with the peak at 530 nm corresponding to the oxidized heme and peak maxima at 522 and 552 nm corresponding to the reduced hemes. (86) 14 2.2.3 Immobilization of PpcA on biomimetic interfaces Recombinant PpcA was immobilized onto a SAM of mixed alkanethiols on a gold electrode as described below. Commercial gold substrates (LG thin films, Santa Clara, CA) were cleaned by dipping in Piranha solution (7 parts sulfuric acid and 3 parts 30% hydrogen peroxide solution) for 30 s and then rinsing with distilled water. For SAM formation, the cleaned substrates were dipped in 5 mM ethanolic solution of 11-mercaptoundecanoic acid and 11mercapto 1 undecanol in a 1:3 ratio for 24 h, and then rinsed with ethanol and distilled water. PpcA was bound to the SAM via an amide linkage using 50 mM 1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 5 mM N-hydroxysuccinimide (NHS) solutions. The electrodes were then washed in 20 mM phosphate buffer to remove unbound protein. 2.2.4 Electrochemical measurements CV experiments were performed using a three cell electrode setup that included the gold substrate described above as the working electrode, a Ag/AgCl reference electrode and a platinum wire counter electrode. Gold electrodes for rotating disk experiments were purchased from Pine Research Instrumentation Inc., Raleigh, NC. All other electrodes were purchased from Bioanalytical Systems Inc, West Lafayette, IN. Electrochemical measurements were conducted using a CHI660B and CHI660D electrochemical potentiostats (CH instruments Inc, Austin TX). Before CV experiments, all solutions were purged with argon gas for a minimum of 30 min to o remove oxygen, and the experiments were performed in an argon atmosphere at 25 C. During the CV experiments, the voltage was alternately swept between 0.2 and -0.8 V (versus Ag/AgCl) at scan rates (scan rates ranging from 20-700 mV/s). The midpoint redox potential of the immobilized PpcA was calculated by averaging the anodic and cathodic peak potentials of CV 15 scan at low scan rate (10mV/s). The midpoint redox potential of PpcA in solution was measured similarly using 1 mg/ml solution of protein in an argon atmosphere. Unless specified otherwise, all experiments were done in presence of 100 mM NaCl as a supporting electrolyte. 2.2.5 Ellipsometric measurements Ellipsometric measurements were carried out using M-44 rotating analyzer ellipsometer ( J.A. Woollan Co. Inc., Lincoln, NE) running WVASE32 software. The refractive index and the extinction coefficient values were assumed to be 1.5 and 0 for all measurements. For immobilized cytochrome c measurements, the film was modeled as a transparent “Cauchy” layer with refractive index of 1.5. A minimum of three measurement were taken for each sample. 2.3 Results and discussion 2.3.1 Production and purification of PpcA SDS PAGE results of the samples taken during fermentation indicated the cytochrome c expression in E. coli. The first step of purification with SP sepharose ® column captured the cationic charged proteins from the periplasmic fraction (Fig. 2.1A). The protein eluted with a step gradient had very high concentration of salt, which may interfere with binding of protein on ion exchange resin. Buffer exchange of the protein samples using gel filtration column reduced the conductivity below 4 ms/cm (Fig. 2.1B). These pooled protein fractions were further purified by second step of cation exchange chromatography with NaCl gradient. The Purified recombinant PpcA obtained by cation exchange chromatography (Fig. 2.2) had a ratio of A210/A408 (peptide bond/heme absorption) near 1, consistent with the incorporation of the protein’s three hemes. (85) The protein samples displayed a single protein band in an SDSPAGE gel that exhibited a relative mass of approximately 9.6 kDa (Fig. 2.2, inset), as reported 16 for the native PpcA protein. (87) The redox difference spectra of dithionite-reduced versus oxidized recombinant PpcA (Fig. 2.3) showed the three absorption bands at wavelengths of 420, 522, and 552 nm, characteristic of c-type cytochromes. 2.3.2 Electrochemistry of PpcA in solution The electrochemical behavior of PpcA was first investigated with protein in solution. The bare gold working electrode contributed to protein degradation during CV runs. Therefore all the electrochemical characterization were done on SAM modified gold electrode. Also presence of oxygen interfered with PpcA electro analysis. Therefore all the measurements were done under the constant stream of argon with buffers purged by nitrogen or argon. The CV curves for recombinant PpcA in solution at 10 mV/s (Fig. 2.4) displayed defined oxidation and reduction peaks that gave a mid-point potential of -375 mV vs. a Ag/AgCl electrode. This value matches closely the midpoint potential of -374.5 mV versus Ag/AgCl (-169.5 mV versus a standard hydrogen electrode) estimated for the native PpcA by cyclic voltammetry at the same scan rate. (88) The peak separation was about 130 mV at scan rate of 100 mV/s and increased with increasing scan rates consistent with quasireversible redox species. The relationship between peak currents and scan rate is give by Randles-Sevcik equation. (84) 𝑖𝑝 = 2.69 ∗ 105 ∗ 𝑛 3� 2 ∗𝐴∗𝐷 1� 2 ∗ 𝐶 ∗ 𝑣 1/2 .................Eq.( 2.1) where, ip is the peak current, n is the number of electrons transferred, A is the area of the electrode, D is the diffusion coefficient, C is the bulk concentration of the analyte and ν is the scan rate. The relationship between ip and square root of ν was linear for alkane terminated 17 SAM indicating diffusion controlled reaction. This relationship was linear for SAM coated gold electrode, indicating diffusion limited current of PpcA at electrode (Fig. 2.5). 2.3.3 Immobilization of PpcA and its electrochemistry o Ellipsometric measurements revealed the monolayers have thickness of 15 A , which is consistent the literature reported height for a 11 carbon chain link with a slight tilt. (89) o Thickness of the SAM after cytochrome c deposition was about 30 A , indicating cytochrome c adsorption in a monolayer. The midpoint potential obtained from CV of immobilized PpcA at a scan rate of 10 mV/s (data not shown) was -372 mV against Ag/AgCl, which is virtually identical to that obtained for PpcA in solution and the native PpcA. (88) Hence, the biomimetic interface with immobilized PpcA is well suited to investigate the electrochemical activity of PpcA under biologically relevant conditions. The CV curves for PpcA immobilized on SAM-functionalized gold collected at increasing scan rates from 25 to 700 mV/s (Fig. 2.6) displayed distinct cathodic and anodic peaks, indicating that the protein was being reversibly oxidized and reduced, respectively. Plotting cathodic peak height for the SAM-immobilized PpcA vs. scan rate (Fig. 2.6, inset) gave 2 a linear correlation (R =0.997), consistent with a surface-controlled electrochemical reaction. (90) The Laviron equation expresses the relationship between peak current Ip, scan rate ν and surface coverage τ: 𝐼𝑝 = 𝑛2 𝐹 2 𝐴𝜏𝜈 4𝑅𝑇 18 ......................Eq.(2.2) where A is area of electrode, n is number of electrons transported, F is Faraday's constant, R is the universal gas constant and T is absolute temperature. Also, 𝐼𝑝 = 𝑛𝐹𝑄𝜐 4𝑅𝑇 ..........................Eq.(2.3) From the area under the peak for a voltammogram, we calculated the charge Q. And from this relationship, we calculated value of n to be 1. From the area under the CV cathodic peak, we 2 calculated PpcA surface coverage values (τ) of 20 to 50 pmol/cm , which are 2-5 times greater than the values reported for other immobilized c-cytochromes, such as horse heart c-cytochrome 2 and cytochrome c 552. (81, 91, 92) This large difference in τ (20 to 50 vs. ~10 pmol/cm ) is surprising given the similar size of the cytochromes (e.g., 9.6 kDa for PpcA vs 12 kDa for horse heart cytochrome) and the same alkane thiols (MUA and MU) being used to make the SAM for both PpcA and horse heart cytochrome c. (27) The difference is likely due to variations of the immobilization methods, suggesting that the immobilization strategy may need to be optimized for each cytochrome to be tested under biologically relevant conditions. Laviron analysis was used to determine the apparent electron transfer rate constant ks for -1 PpcA immobilized on a gold electrode. Laviron’s calculations for the variation of parameter m (m = (RT/F)*(k/nν)) with peak potential separation (ΔEp) were fitted to a third order polynomial, -1 which was then used to determine the values of m experimental ΔEp results. 19 at different sweep rates from the 𝑅𝑇 ∆𝐸𝑝,𝑎 = (1−𝛼)𝑛𝐹 𝑙𝑛 � ∴ ∆𝐸𝑝,𝑎 = 𝑅𝑇 𝑙𝑛(1 (1−𝛼)𝑛𝐹 (1−𝛼) � 𝑚 ...................Eq. (2.4) 𝑅𝑇 − 𝛼) + (1−𝛼)𝑛𝐹 𝑙𝑛(𝑚−1 )..............Eq. (2.5) Where, α is a transfer coefficient. Plot of experimental Δ Epa vs ln(m-1) and Δ Epc vs. ln (m-1) can be used to calculate the ks values (Fig. 2.7). 𝑅𝑇 ∆𝐸𝑝,𝑎 = (1−𝛼)𝑛𝐹 𝑙𝑛 � where, 𝑚= (1−𝛼)𝑛𝐹 𝜗 𝑅𝑇 𝑅𝑇𝑘 𝑜 𝑛𝐹𝜗 𝑘𝑜 � ..................Eq. (2.6) .......................Eq. (2.7) Similarly, ∆𝐸𝑝,𝑐 = − 𝑅𝑇 𝛼𝑛𝐹 𝑙𝑛 � 𝛼𝑛𝐹 𝜗 𝑅𝑇 𝑘 𝑜 �..................Eq. (2.8) -1 Fitting the equations 2.6 and 2.8, the ks for reaction was found to be 0.05 s . These results indicate relatively rapid ET between immobilized PpcA and the electrode. In summary, we have successfully expressed and purified Geobacter periplasmic cytochrome c PpcA in E. coli. The heterologously expressed PpcA is electroactive and have redox properties similar to the native Geobacter cytochrome. The PpcA protein is capable of direct electron transfer with electrode. Furthermore, we have successfully immobilized the PpcA protein on SAM covered gold electrode and studied the redox properties of the immobilized protein. This work is an important step in developing biomimetic redox active cell envelope or 20 BRACE interface on the electrode, where we need the protein to be able to interact with the electrode in a controlled fashion. 21 3.0 ELECTROCHEMICAL CHARACTERIZATION OF GEOBACTER SULFURREDUCENS PPCA C-CYTOCHROME USING BIOMIMETIC INTERFACES REVEALS GREATER REDUCTION RATES OF URANIUM THAN IRON 3.1 Abstract The reductive precipitation of uranium by Geobacter bacteria shows promise for the in situ bioremediation of contaminated sites. However, genetic approaches to identify the redox components involved in the reaction are often limited by compensatory effects of the mutations, which often lead to adapted phenotypes. To bypass this limitation we developed nanostructured biomimetic interfaces that mimic the cell envelope of Geobacter cells and permit the interrogation of specific redox components. We used this approach to investigate the electrochemical role of the most abundant periplasmic Geobacter c-cytochrome, PpcA, in uranium reduction. The studies revealed a much greater rate constant of PpcA for the reduction of uranium than for iron electron acceptors, providing a plausible explanation for the rapid reduction and precipitation of uranium, and loss of cell viability, observed when the radionuclide permeates into the periplasm. As a result, Geobacter cells have evolved mechanisms for the extracellular reduction of uranium to prevent it from traversing the outer membrane. The studies highlight the value of biomimetic interfaces to interrogate individual redox components of the Geobacter cell envelope and gain novel insights into their biological role, and also show promise for the development of sensors and deployable devices for uranium bioremediation. 3.2 Introduction Uranium (U) is naturally present at low levels in many terrestrial environments and can reach higher concentrations in seawater and U-rich ores. (93) Its anthropogenic use in nuclear research, fuel production, coal combustion, phosphate fertilizers, and weapons manufacturing has also 22 resulted in the accumulation of toxic levels of the radionuclide in many soils and groundwater. (94) In these environments, U is easily oxidized into the water-soluble and highly mobile uranyl +2 cation (UO2 ), which facilitates its dispersal. This results in vast volumes of groundwater contaminated with U in complex mixtures with other radionuclides and toxic metals, making environmental remediation via standard excavation-and-removal or pump-and-treat approaches impractical. (95) Despite its toxicity even at trace concentrations, (96) some microorganisms inhabit environments with naturally high U content and contribute to its cycling. (97) Among these microorganisms are dissimilatory metal reducing bacteria in the family Geobacteraceae, which can gain energy for growth by transferring respiratory electrons to the uranyl cation. (98, 99) The biological reduction of the hexavalent U (U[VI]) in the uranyl cation by these organisms generates sparingly soluble phases of tetravalent U (U[IV]), (99-101) thereby reducing the mobility and bioavailability of the contaminant. Furthermore, the reductive precipitation of U by these microorganisms can also be stimulated in situ with field-scale additions of selected electron donors such as acetate, (102, 103) thus slowing the migration of the contaminant and mitigating health risks associated with U exposure. Studies with the model representative Geobacter sulfurreducens show that the reduction of U by these organisms is extracellular and dependent on the expression of conductive protein filaments or pili and outer membrane c-cytochromes. (100) The contribution of outer membrane c-cytochromes is likely small, because protease treatment to remove proteins exposed on the outer membrane does not affect U reduction. (60) Furthermore, the extent of U reduction correlates linearly with the piliation levels (100) but not with the outer membrane c-cytochrome content. (104) 23 Some U can also traverse the outer membrane of Geobacter cells, especially if non-piliated. (100, 104) Once inside the periplasmic space, U is rapidly reduced to a mononuclear U(IV) phase bound to carbon ligands consistent with the involvement of periplasmic proteins. (100) As the degree of periplasmic precipitation increases, the cellular respiratory activities decline rapidly and the cell’s viability is severely compromised. (100) The periplasm of Geobacter cells contains abundant small c-cytochromes, which could catalyze this reduction and influence the cells’ ability to remain viable during exposure to U. One such periplasmic protein is PpcA, (88) a triheme c-cytochrome that is conserved in all the sequenced Geobacteraceae genomes. (56, 105) PpcA has been proposed to function as a periplasmic electron carrier during acetate oxidation − + + (88) and to contribute to the H electrochemical potential gradient through a coupled e /H transfer mechanism. (106) However, the role of PpcA in U reduction remains controversial. Resting cell suspensions of a PpcA-deficient mutant were originally reported to be impaired in the acetate-dependent reduction of U(VI) but were not affected when hydrogen served as the electron donor. (88) However, later studies failed to replicate the acetate-dependent mutant phenotype. (107) Such contrasting results could have been the result of compensatory effects of the mutation, as reported for other c-cytochromes in Geobacter. (108, 109) To bypass these limitations, we studied the catalytic activities of PpcA in a controlled in vitro environment using electrochemical interfaces structured to mimic the native cell envelope. Cyclic voltammetry was used to investigate the electron transfer (ET) kinetics of PpcA to Fe(III) and U(VI), provided as soluble complexes with various ligands. The studies revealed much higher rate constants for U(VI) than for Fe(III) independently of the ligand, consistent with a much higher affinity for U than for the natural electron acceptor Fe. The results provide novel insights into the role of PpcA in U reduction that are relevant for the in situ U bioremediation. 24 3.3 Materials and Methods 3.3.1 Recombinant PpcA expression and purification. PpcA was heterologously expressed in Escherichia coli strain BL21 DE3, as reported in chapter 2. 3.3.2 Biomimetic interfaces with PpcA. The conceptual design for the biomimetic interfaces constructed and interrogated in this work includes a gold electrode, a self-assembled monolayer of alkanethiols (SAM), a PpcA monolayer, and, when indicated, an external synthetic lipid bilayer. The conceptual design is shown in the Figure 3.1, where the SAM and PpcA layers serve as mimics of the inner membrane (IM) and periplasm space (PS), respectively, and the external, synthetic lipid bilayer, as the outer membrane (OM) analogue. PpcA was immobilized onto SAM coated gold electrode as described in chapter 2. When indicated, a synthetic lipid bilayer was deposited by incubating the PpcA-containing SAM interface with a 0.25 mM solution of 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) liposomes in 50 mM HEPES buffer. The resulting interface, designated DOPC interface, mimicked the cell envelope of G. sulfurreducens, with its PpcA-containing periplasmic space sealed by an outer membrane. 3.3.3 Electrochemical measurements. Cyclic voltammetry (CV) experiments were performed using a three-electrode setup consisting of the PpcA-functionalized gold substrate described above as the working electrode, a Ag/AgCl reference electrode, and a platinum wire counter electrode. All the electrodes were purchased from Bioanalytical Systems Inc (West Lafayette, IN), except for rotating disk electrodes (RDE), which were purchased from Pine Research Instrumentation Inc. (Raleigh, 25 NC). Prior to SAM deposition, the RDE were polished with alumina slurries (1, 0.3, and 0.05 micron grade) on polishing pads. Electrochemical measurements were conducted using a CHI660B electrochemical potentiostat (CH instruments Inc, Austin TX) and, for RDE experiments, a modulated speed disk electrode rotator (Pine Research Instrumentation Inc., Raleigh, NC). Prior to CV, all solutions were purged with argon gas for a minimum of 30 min to o remove oxygen, and the experiments were performed in an argon atmosphere at 25 C. During the CV experiments, the voltage was alternately swept between 0.2 and -0.8 V (versus Ag/AgCl) at scan rates (ν) ranging from 25-700 mV/s. Unless specified otherwise, the supporting electrolyte in all the experiments was 100 mM NaCl. When indicated, CV of the PpcA or DOPC interfaces was performed in the presence of various concentrations of the following soluble, chelated forms of iron (Fe) and uranium (U): ferric nitrilotriacetate (Fe-NTA), ferric citrate (Fecitrate), ferric nitrate (Fe-nitrate), uranyl acetate (U-acetate), and uranyl nitrate (U-nitrate). Rotating Disc Electrode Voltammetry (RDE): The second order rate constant ko' values were also validated using the RDE technique, which allows the interface mass transfer limitations to be controlled. The PpcA interface was constructed in the same way as described for the static cyclic voltammetry studies but using a circular (5-mm diameter) gold electrode (Pine Research Instruments) and attached to the rotating shaft of the RDE apparatus. The stability of the PpcA interface was first investigated using variable speeds to determine the maximum rotation speed (2500 rpm) it withstood. Then, Fe species were added at various concentrations to collect the voltammograms. Fig. 3.5 shows a representative rotating disc voltammogram for Fe-NTA. For all the RDE experiments, the potential of the working electrode was maintained at -500 mV against Ag/AgCl. The steady state 26 current (iss) was measured for a range of rotation speeds (ω) and the inverse of the steady state -1/2 current values (1/iss) were plotted against ω . Fig. 3.6A shows the Fe-NTA plot, as an example. The limiting current ilim was inferred from the Y axis intercept value of the regression line for each electron acceptor concentration. This value was plotted against the electron acceptor concentration to generate a regression line whose slope corresponds to the second order rate constant ko’. The ilim values follow a linear correlation with the concentration of the electron acceptors. Fig. 3.6B shows the linear plot for Fe NTA, as an example. The linearity of the correlation can be used to calculate the ko’values as previously described (110) using the equation: 𝑖𝑙𝑖𝑚 = 𝑛𝐹𝐴𝐶𝜏𝑘𝑜′ ......................................(Eq. 3.1) where n is the number of electrons transferred, F is Faraday’s constant, A is the area of electrode, C is the concentrations of the electron acceptor, and τ is the PpcA surface coverage. Calculation of rate constants: The reduction of a soluble electron acceptor by PpcA immobilized on the SAMfunctionalized gold electrode is a two-step reaction that first involves reduction of PpcA by the electrode and then the reduction of the electron acceptor by PpcA, as exemplified in the following reactions: 𝑃𝑝𝑐𝐴𝑂 + 𝑒 − ⇆ 𝑃𝑝𝑐𝐴𝑅 (PpcA reduction by gold electrode) 𝑘o' 𝑃𝑝𝑐𝐴𝑅 + 𝑀𝑂 → 𝑃𝑝𝑐𝐴𝑂 + 𝑀𝑅 (PpcA oxidation by electron acceptor) 27 where PpcAO and PpcAR are the oxidized and reduced forms of PpcA, respectively, and MO and MR are the oxidized and reduced forms of the soluble electron acceptor, respectively. The second order rate constant for reduction of MO by PpcAR is ko’. To calculate ko’, we performed CV on PpcA immobilized on SAM-functionalized gold electrodes at scan rates of 25-750 mV/s in the presence or absence of the soluble electron acceptors, provided at various concentrations, as indicated for each experiment. The CV curves were normalized by subtraction of the background current recorded with phosphate buffer on SAM coated electrode in potential sweep voltammetry. (111) The peak current values (ip) were measured for the PpcA interface in the absence of electron acceptors and peak potential values (Ep) were noted. The catalytic current values (ik) were measured at the Ep in the presence of the soluble electron acceptors. From the ratio of ip to ik, we determined the kinetic parameter ϕ using the working relationship given by Nicholson and Shain for catalytic reactions. (90) The ϕ values obtained for each of the catalytic reactions involving a particular electron acceptor were plotted o versus the reverse of the scan rate (1/υ). The slope of the linear regression line, designated K , o was used to calculate the Kf constant using Eq. 1: 𝐾𝑓𝑜 = 𝐾 𝑜 𝑛𝐹�𝑅𝑇 .......................................(Eq. 3.2) where n is the number of electrons transferred, F is Faraday's constant, R is the universal o gas constant, and T is the absolute temperature. The Kf value obtained from Eq. 1 was plotted 28 against the electron acceptor concentration and the slope of the regression line corresponded to ko’. We measured the second order rate constant in triplicate independent experiments for each electron acceptor. In addition, second order rate constants for Fe species were also measured by rotating disc voltammetry. Pairwise comparisons between the electron acceptor rate constants were statistically analyzed using the t-test function of the Microsoft  Excel  software. Statistically significant data sets (p < 0.05 or < 0.005) were indicated with one or two star symbols, respectively. 3.4 Results and Discussion 3.4.1 Catalytic activity of PpcA with iron and uranium salts. The reduction of soluble Fe and U species by PpcA was investigated in the PpcA biomimetic interfaces (Fig. 3.2). Control experiments performed with the PpcA interface in the absence of soluble metal species resulted in Nernstian CV curves with well-defined, reversible peaks, indicating that PpcA was engaging in one-step redox reactions with the gold electrode. Addition of Fe-NTA (Fig. 3.2A) or U-acetate (Fig. 3.2B) to the PpcA interface increased the cathodic current, but no anodic peaks were detected, as expected of an irreversible reductive reaction. The addition of the soluble electron acceptors also resulted in sigmoidal CV curves typical of two-step electron transfer reactions, (90) which in this case involved the electron transfer from the electrode to PpcA and then from PpcA to the electron acceptor. Furthermore, the cathodic current of the PpcA interface increased with increasing electron acceptor concentration. By contrast, control interfaces of SAM-functionalized gold electrodes without PpcA lacked faradic current even when in the presence Fe-NTA or U-acetate (Fig. 3.2A and 3.2 B, respectively). Hence, the SAM layer effectively blocked access of the soluble electron 29 acceptors to the underlying gold electrode, and any cathodic current detected by CV was due to electron transfer via PpcA. In the native environment, PpcA is trapped in the periplasmic space between the inner and outer lipid membranes. We mimicked this architecture by depositing 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) liposomes on the PpcA biomimetic interface and form a lipid bilayer (112, 113) that completely covered the interface. The addition of the DOPC lipid bilayer prevented the increases in the PpcA catalytic current observed in the presence of Fe-NTA or Uacetate, and the CV curves only exhibited the peaks corresponding to the transfer of electrons between the gold electrode and the PpcA (Fig. 3.2, insets). Hence, the synthetic lipid bilayer prevented the permeation of the soluble metal species inside the periplasmic mimic and thereby blocked their access to PpcA. These results are particularly significant to understand how metals transverse the outer membrane and are reduced in the periplasm of Geobacter cells. Our results suggest, for example, that the outer membrane functions as a mechanical barrier for metal ions, effectively preventing them from reaching the periplasmic space. However, in the biological setting, the outer membrane contains porins and transporters, which facilitate the diffusion and/or transport of soluble species and allow metals and metal chelators into the periplasm. One of the most abundant proteins in the G. sulfurreducens outer membrane is, for example, a putative 3+ porin that is required for the reduction of soluble, chelated forms of Fe . (114) This porin may allow soluble metal species to enter the periplasm, where they can be reduced by periplasmic ccytochromes such as PpcA. 30 ’ 3.4.2 PpcA exhibits high second order rate constants (ko ) for the reduction of U. The electrochemical studies of PpcA reduction in the biomimetic interfaces allowed us to calculate the second order rate constant (ko’) for the reduction of various Fe and U electron acceptors. The calculations used the Nicholson and Shain model, (90) which is based on the following assumptions (115, 116) : (i) the reaction of PpcA with the electrode is a fast, reversible, one-electron transfer reaction (as we validated experimentally); (ii) the reaction between PpcA and the soluble electron acceptor follows pseudo first order kinetics and is irreversible (which we showed under the experimental conditions, Fig. 3. 2); (iii) no adsorption of products or reactants occurs; and (iv) the diffusion coefficients of all species are the same. From the CV curves recorded at various scan rates for the PpcA interface alone or in the presence of different electron acceptor concentrations (Fe-NTA, Fe-citrate, Fe-nitrate, U-acetate, and U-nitrate), we calculated the ratio of ik to ip, and then the kinetic parameter ϕ. (90) This kinetic parameter relates the ratio of ik to ip with the reaction rates as described in the case of catalytic reaction with reversible charge transfer. (90) Provided all the assumptions of the Nicholson and Shain model are approximately met, the ϕ versus 1/ υ (reverse of scan rate) plot is expected to yield a straight line. We did obtain linear correlations for all the electron acceptors but only at scan rates of 300 mV/s or higher. For example, the correlation between ϕ and 1/ υ was linear for Fe-NTA concentrations above 800 µM (Fig. 3.3A). This suggests that some of the model’s assumptions are not met at the lowest scan rates. The Nicholson and Shain model was developed for soluble species under the assumption that all reactants and products had the same diffusion coefficient. (90) In our system, where PpcA is immobilized on the SAM-functionalized electrode, this assumption is only valid 31 at higher scan rates, at which the reactant concentration profiles at the surface is only partially developed, and the observed rate depends solely on the reaction kinetics. However, at lower scan rates, the electrochemical measurements are influenced by not only reaction kinetics but also by the diffusion of the reactant to the surface. From the slope of the regression line within the linear region of the ϕ versus 1/ υ, we o calculated the K parameter and used Eq. 3.2, described in the Materials and Methods section, to o o calculate the Kf constant. From the linearity of the plot of Kf versus the concentration of the electron acceptor, we calculated the second order rate constant ko’ for the reduction of various Fe and U chemical species by PpcA (Table 3.1). Fig. 3.3 B shows, as an example, the plot used to estimate the second order rate constant ko’ for Fe-NTA. Pairwise comparisons between FeNTA and the other Fe species (Fe-citrate and Fe-nitrate) revealed significant variations in the rates of reduction (Fe-nitrate > Fe-NTA > Fe-citrate) (Fig. 3.4). Similar results were obtained by rotating disc electrode voltammetry (Table 3.1), suggesting that the differences in the rates of reduction were not an artifact of the electrochemical measurements. Rather, the chemical ligand of Fe influenced the rates of reduction. This result is in agreement with kinetic studies of ccytochromes from the metal-reducing bacterium Shewanella oneidensis, which also reduce Fe(III) species at rates that are influenced by the type of ligand complexed to the metal. (117) The charge and molecular size of the ligand affects the kinetics of binding to the cytochrome and also the position and distance between metal electron acceptor and the heme Fe of the protein. This, in turn, influences the rates of electron transfer. 32 Interestingly, the ko’ values for the reduction of the U species (U-acetate and U-nitrate) were significantly higher than for the Fe species (Fig. 3.4). On average, the rates of reduction of Fe species were 0.06 (± 0.04), whereas those for U species were more than 10 times greater (0.79 ± 0.01). Such differences cannot be attributed to the type of ligand because the reduction rates of U-nitrate were, for example, 8-fold greater than for Fe-nitrate (p = 0.003), although the two electron acceptors have the same ligand. Hence, the faster kinetics of the reduction of U electron acceptors by PpcA is likely the consequence of increased thermodynamic driving force for U reduction over Fe and/or differences in protein-metal interactions. 3.4.3 Specific interactions between U and PpcA promote reduction rates. The parameters that influence the rate constant (ket) for electron transfer between donor and acceptor can be interpreted using semiclassical theory, (118-120) as shown in Eq. 3.3: 𝑘𝑒𝑡 = � 4𝜋3 ℎ2 𝜆𝑘𝐵 𝑇 2 𝐻𝐴𝐵 𝑒𝑥𝑝 �− (∆𝐺 0 + 𝜆)2 4𝜆𝑘𝐵 𝑇 � ..............................(Eq. 3.3) 0 where kB is Boltzmann’s constant, h is Plank's constant, ∆G is the free energy of the reaction, HAB is the electronic coupling matrix element, and λ is the nuclear reorganization energy. The rates of redox reactions involving metals are often proportional to the 0 thermodynamic driving force, ∆G , (121) which is dependent on the reduction potentials of the oxidized/reduced metal pair and, to a lesser extent, on the type ligand complexation through the o reaction’s electrochemical potential difference (∆E ). (117) To investigate a similar influence in 33 0 the PpcA-mediated reactions we used Eq. 3.4 to calculate ∆G for the various U and Fe electron acceptors tested. ∆𝐺 𝑜 = −𝑛𝐹(∆𝐸 𝑜 ) ............................(Eq. 3.4) To minimize the ligand’s contribution, we focused on the reduction of the U-nitrate and Fe-nitrate, which share the same ligand (nitrate). The standard redox potential (E°) of the half 2+ reaction of UO2 , as shown below, results in a free energy value of -87 kJ/mol. 𝑈𝑂22+ + 2𝑒 − + 4𝐻 + → 𝑈 4+ + 2𝐻2 𝑂, 𝐸 0 = 280 𝑚𝑉 𝑣𝑠. 𝑆𝐻𝐸 For the half reaction of Fe3+, shown below, we calculated a free energy of -91 kJ/mol. 𝐹𝑒 3+ + 𝑒 − → 𝐹𝑒 2+ , 𝐸 0 = 771 𝑚𝑉 𝑣𝑠. 𝑆𝐻𝐸 The free energy of the two reactions is remarkably similar, failing to explain the greater rates of U-nitrate reduction by PpcA compared to Fe nitrate determined experimentally (Fig. 3.4). 0 With similar ∆G values for Fe and U acceptors, only larger donor-acceptor electronic couplings, HAB, and/or lower reorganization energies, λ, can explain the greater reaction rates of the PpcA-U pair compared to PpcA-Fe. Both of these variables are intimately connected with the type of coordination between PpcA and the metal acceptor. Prior to electron transfer, the metal forms a precursor complex, which positions the metal in a fashion that influences the rates of electron transfer. The HAB variable indicates the probability of electron transfer in this metalprotein precursor complex upon thermal activation. (122) Cytochromes can have many sites for 34 binding the metal electron acceptor, thus forming several precursor complexes in which the donor-acceptor distance and bridge varies. (123) The electronic coupling matrix element and the rates of electron transfer decrease, for example, exponentially with donor-acceptor distance. Hence, greater rates of U reduction could result from PpcA coordinations that reduce the distance between the U acceptor and the electron-donating heme group in the cytochrome. The type of PpcA coordination around the metal acceptor can also have substantial effects on λ, which is the energy required to distort the nuclear configuration of the reactants into the equilibrium configuration of the products prior to electron transfer. The stoichiometry, size, charge, and structural properties of the reacting protein donor and metal acceptor have a profound effect on both the external (outer sphere, λo) and internal (inner sphere, λi) reorganization energy required to reach the equilibrium configuration of the products. (124) The outer sphere relates to the surrounding medium and refers to changes in the protein matrix and the energy required to rearrange the solvent environment. On the other hand, the inner sphere is influenced by the solvent sheath and the type of ligands involved in creating a geometrically favorable arrangement between the metal and the electron-donating heme group in PpcA prior to electron transfer; hence, it is influenced by redox-dependent nuclear perturbations of redox centers, such as changes in bond angles and lengths. Extended X-ray Absorption Fine Structure (EXAFS) analyses of the uranyl coordination in wild-type cells of G. sulfurreducens with a predominantly periplasmic precipitation (e.g., those not expressing conductive pili) shows U coordinated with carbon-containing ligands. (100) The EXAFS model is consistent with a U atomic environment involving bidentate and monodentate carbon-containing ligands. Bidentate carbon ligands have been reported for other iron-binding proteins such as apotransferrin, which uses the carboxyl coordinations of acidic 35 amino acids as metal ligands. (125) The uranyl cation also binds to apotransferrin at its ironbinding sites, using some of the amino acids of the Fe coordination sphere as well as specific amino acid ligands. (126) Interestingly, the binding of the uranyl cation to apotransferrin induces conformational changes such that the period of conformation stabilization is reduced compared to Fe. (126) Similar effects could allow PpcA to position the uranyl cation more rapidly than iron in an optimal configuration for electron transfer, thereby increasing the rates of electron transfer to U acceptors over Fe. 3.5 Implications The rates of electron transfer between cytochromes and metal acceptors reflect the contribution of three rate constants, each corresponding to reactions occurring before, during, and after the transfer of electrons from the protein to the metal, as follows: 𝑘1 Cyt + M ⇔ [Cyt M] 𝑘et [Cyt M] ⇔ [Cyt + + M − ] 𝑘𝑑 [Cyt + + M − ] ⇔ Cyt + + M − The first step involves the diffusion of the metal species (M) to the cytochrome (Cyt) and their association to form a precursor complex [CytM] (rate constant k1). The second step is the + - transfer of electrons from the Cyt to the M and formation of the reactant complex [Cyt + M ] + - (rate constant ket). The final step is the dissociation of the [Cyt + M ] complex to generate the + - products Cyt and M , which diffuse apart in the solution (rate constant kd). Calculation the 36 overall reaction rate constant, kobs, experimentally and/or by deconvolution of ket from other rate constants is challenging. (86) However, the biomimetic interfaces used in this study provide a well-characterized and reproducible experimental tool to study redox catalyses involving cytochromes despite the inherent complexity of the stepwise reaction. A major advantage of biomimetic interfaces over the natural, biological system is that the former allows virtually all variables (cytochrome content, lipid content, etc.) to be precisely known and controlled to test hypotheses about how each variable influences kobs. The electron transfer rates measured for PpcA under identical conditions but using different metal species revealed, for example, a higher rate constant for U(VI) reduction than for Fe(III) reduction by PpcA. This finding helps explain why U is rapidly reduced in the periplasm of G. sulfurreducens once it transverses the outer membrane. (2, 41, 127) The reduction of U(VI) to U(IV) precipitates the radionuclide as a mineral in the periplasmic space, compromising the structural integrity and essential functions of the cell envelope and eventually killing the bacteria. (100) Not surprisingly, Geobacter cells have evolved mechanisms to reduce the uranium extracellularly and, in doing so, preserving the cell envelope’s essential functions. Another advantage of biomimetic interfaces is that additional redox components of the Geobacter respiratory machinery could be added to extend the kinetic and thermodynamic studies and provide additional insights into bacterial mechanisms for U reduction. This approach could lead to new or improved schemes for the in situ remediation of U and other metal contaminants. The higher reaction rate constant for PpcA-mediated reduction of U than of Fe, independently of its complexation with ligands, also shows promise for the developing PpcAbased electrochemical devices for the specific detection of U, providing a valuable tool for the in 37 situ monitoring of U levels in sites undergoing environmental restoration or for long-term stewardship of contaminated sites. 38 4.0 HETEROLOGOUS EXPRESSION OF OMCB IN E. COLI AND ITS LOCALIZATION TO BACTERIAL MEMBRANE 4.1 Abstract Outer membrane cytochromes play a key role in metal-respiring bacteria like Geobacter and Shewanella spp. The outer membrane cytochrome OmcB of Geobacter sulfurreducens is potentially important protein in Geobacter, although it's primary function in electron transport chain remains ambiguous. Very little information is available about the structure and activity of this protein, in part due to difficulty in isolating active OmcB from Geobacter membranes and the lack of heterologous expression systems for OmcB. In this paper, we report heterologous expression of OmcB in E. coli. Subcellular fractionation localized the protein in E. coli outer membrane, and whole cell spectroscopy confirms redox activity of the heterologously expressed OmcB. 4.2 Introduction Dissimilatory metal reducing microorganisms generating energy in form of ATP through a respiratory process that transfers electrons obtained from intracellular electron donors (commonly organic acids, such as acetate) to extracellular electron acceptors (commonly metal oxides, such as Fe2O3). The electrons are shuttled through an electron-transport chain that includes both soluble and membrane-bound cytochromes. (6, 128, 129) Metal-reducing microorganisms have utility in bioremediation (2, 130) and microbial fuel cells. (131-133) Members of the genus Geobacter have been extensively studied because of their exceptional electrogenic capacity. Proteins involved in Geobacter’s electron transport chain are being isolated and characterized to elucidate the metal-reduction mechanisms. The Geobacter cells contain multiple outer-membrane c type cytochromes that are believed to participate in 39 extracellular electron transfer (ET), either directly or indirectly. (34) Although a large number of these cytochromes have been identified and implicated in their role in extracellular ET, very few of them been studied in detail. To date, OmcZ (134) and OmcS (20) are the only Geobacter outer membrane cytochromes that have been purified and studied in isolation. The lack of high yield system for expressing these proteins is one of the underlying factors. OmcB is a multiheme (12 heme-binding domains) protein partially embedded in Geobacter outer membranes. (27) Unlike other c-type cytochromes that are loosely bound to the outer membrane, OmcB is very tightly associated with the outer membrane and is only partially exposed. (135) Geobacter cells with OmcB deleted were unable to reduce soluble as well as insoluble Fe(III) (27), which may indicate OmcB’s importance in iron reduction. However, Geobacter cells with OmcB deletion mutation, adapted to grow over time (39), suggesting a compensatory mechanism, in which Geobacter upregulates production of other membrane c type cytochromes like OmcS and OmcT. (39, 136) Such pleiotropic compensation may explain the ability of Geobacter with OmcB deletion to reduce uranium (15) as well as to produce electricity (137) Pleiotropic effects also complicate genetic approaches to identify key electron carriers in Geobacter’s native uranium reductase machinery. For example, deletion of OmcF gene also led to reduced expression of OmcB protein, and resultant cells showed impaired growth on Fe (III) citrate. (37) Absence of OmcG and OmcH, did not affect the transcript levels of OmcB; however, expression of OmcB was affected negatively in those strains. (38) Deletion of OmcB led to reduced growth of the cells, but cells eventually recovered with increased expression of other outer membrane cytochromes like OmcS and OmcT. (39) These pleiotropic effects can be bypassed through the study of isolated protein(s) of interest using a biomimetic approach, in which only the protein(s) of interest are assembled into an in vitro interface designed to mimic portions of the Geobacter 40 electron transport chain. Toward that end, heterologous expression of OmcB in E. coli could enable large amounts of the protein to be produced for study. Moreover, because E. coli does not produce any outer membrane cytochromes, heterologous expression of an outer membrane cytochrome would enable the protein’s activity to characterized while the protein was embedded in the outer membrane without interference by other cytochromes. This capability is particularly valuable for outer membrane cytochromes like OmcB that are difficult to isolate and purify in an active state. Here we report for the first time recombinant expression of OmcB cytochrome in E. coli, confirmation of its identity and evidence of its localization to outer membrane of bacteria. 4.3 Methods 4.3.1 Sequence data To express C-terminal His6-tagged OmcB in E. coli, the signal peptide of G. sulfurreducens OmcB was replaced with that of E. coli OmpA. The primers OmpA-OmcB 5' NdeI (5’- ACCATATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCG TAGCGCAGGCCTGCGGCTCCGAAAACAAGGA-3’, coding sequences of E. coli OmpA signal peptide are underlined), and OmcB XhoI 3’ His fusion (5’- GTAAGTCTCGAGCGGACGGGTCGTGCCGAGGTA-3’) were used for PCR to amplify the omcB gene. The PCR product was digested with NdeI and XhoI and cloned into the NdeI- XhoI site of pET21c vector (Novagen), yielding pET21cOmpAOmcB. Plasmid pET21cOmpAOmcB was used as template to substitute the cysteine residue right after signal peptide to alanine by mutagenesis PCR using two primers OmcB Cys mut 5’ (5’- GTAGCGCAGGCCGCCGGCTCCGAAAAC-3’, mutated sequences are underlined.) and OmcB Cys mut 3’ (5’-GTTTTCGGAGCCGGCGGCCTGCGCTAC-3’, mutated sequences are 41 underlined.), yielding pET21cOmpAOmcB24. The AATACCATGGGCTCCGAAAACAAGGAGGGGAC-3’) primers and OmcB OmcB 5’ 3’ NcoI (5’- XhoI (5’- ATATCTCGAGCGGACGGGTCGTGCCGAGGTAG-3’) were used to clone pET22bOmcB that expresses mature OmcB fused to E. coli PelB leader sequence and His6-tag at the N- and Cterminal, respectively. The integrity of each plasmid was confirmed by sequencing analysis. Primers used for cloning of OmcB are listed in Table 1. 4.3.2 Bacterial strains and plasmids E. coli strain DH5α (Invitrogen) was used for subcloning and strain BL21 (DE3) (Novagen) was used for heterologous expression of protein with and without His tag. E. coli cells (BL 21 DE3) were freshly co-transformed with plasmid PEC 86 containing ccm gene which is responsible for the heme maturation of cytochrome c as well as OmcB plasmids. The cells were selected, grown, and maintained into 2X YT media (16 g/L Bacto Tryptone, 10 g/L Yeast extract, 5 g/L NaCl) containing selective antibiotics: chloramphenicol (34 µg/ml) and ampicillin (100 µg/ml). For protein expression a single colony was picked from plate and grown into 50 ml 2X YT media at 30o C overnight. 10 ml of this culture was used to inoculate 1L M9 minimal media supplemented with casamino acids (10 ml of 10% solution), vitamin B1 (100 µL of 0.5%), magnesium sulfate (1 ml of 1 M stock solution), glucose (10 ml of 20% solution) and antibiotics o chloramphenicol (34 µg/ml) and ampicillin (100 µg/ml). The culture was incubated at 30 C while shaking at 225 rpm until the optical density (O.D.) reached around 0.8, and the cells were induced with isopropyl-1-thio-b-D-galactopyranoside (0.1 mM), and were supplemented with heme (10 µM final from filter sterilized stock of 5 mM) and δ-aminolevulinic acid (0.3 mM). After induction the shaking was reduced to 190 rpm, and cells were grown for additional 15-18 42 hr before harvesting. In some experiments the induction step was omitted. The 2X YT and LB media were also used at different temperatures and shaking conditions to optimize growth parameters. Samples from the fermentation media were taken periodically, and, after reading optical density at 600 nm, the samples were centrifuged, and digested by boiling with Laemmli buffer containing 2.1 % SDS (100 µl of buffer per 0.1 O.D. reading). The hexahistidine (His) affinity tag was added to the c-terminus of protein to aid affinity purification of the protein. The NcoI-XhoI restriction fragment of OmcB was cloned into vector pET-22b(+), which contains pelB leader sequence to facilitate the transport of OmcB polypeptide into the periplasmic space where further posttranslational processing takes place. (49, 53) The OmpA tag of the pET21cOmpAOmcB also targets proteins to the periplasm. Both of the plasmids are under the control of T7lac promoter. The plasmid pEC86 allowed constitutive expression of ccm genes (genes required for cytochrome c maturation) under the aerobic conditions.(55) Expression of OmcB was optimized by adjusting variables including temperature, shaking speed and induction time and concentration of the inducer IPTG. Color of the cell pellet (pink for the cells producing mature cytochrome c) and spectroscopic analysis of cells reduction by sodium dithionite served as a preliminary indication of mature cytochrome c production. SDS PAGE analysis of the whole cells digested by heating with SDS buffer revealed a band of OmcB at ~80 kD which was absent in the control cells without the OmcB plasmids. From the ratio of the cytochrome c peak at 552 nm to the intensity of the band at ~80 KD in SDS PAGE, the optimal fermentation conditions for OmcB expression were evaluated. 43 4.3.3 Subcellular fractionation Subcellular fractions were prepared using a procedure modified from the previously described methods. (11, 138-140) Briefly, the protein expression cultures were harvested by o centrifugation at 6000 X g at 4 C for 20 min. The harvested cells were washed with 40 ml cell resuspension buffer (50 mM Tris HCl pH 8, 100 mM NaCl, 10 mM MgCl2 and 10 µg/ml DNase). The clarified cell expression media at the end fermentation as well as cell resuspension buffer after cell wash were concentrated using an ultrafilter with molecular weight cutoff of 10 kD, and samples were stored for further analysis. The washed cells were pelleted by o centrifugation for 10 min at 6000 X g at 4 C and suspended in 100 mM Tris HCl pH 8 followed by addition of sucrose (75 mM), EDTA (50 mM) and lysozyme (0.1 mg/ml) which leads to o spheroplast formation. The spheroplasts were separated by centrifugation at 20000 X g at 4 C for 20 min. The supernatant was stored as a periplasmic fraction. The spheroplasts were suspended in resuspension buffer and lysed by Avestin Emulsiflex C3 high pressure homogenizer by 3 passes at 12000 psi. The unbroken cells were separated by centrifugation at o o 4000 X g at 4 C for 20 min, and the supernatant was ultracentrifuged at 100000 X g at 4 C for 90 min in a Beckman Ti 45 rotor. The supernatant was stored as a cytoplasmic fraction, and the pellet was stored as a membrane pellet. The membrane pellet was resuspended in cytoplasmic membrane solubilization buffer containing 50 mM Tris HCl, pH 8, 100 mM NaCl and 1 % o sarkosyl for 30 min. The membrane suspension was ultracentrifuged at 100000 X g at 4 C for 60 min in Beckman Ti 45 rotor. The supernatant was stored as inner membrane fraction (IM), and the pellet was stored as outer membrane fraction (OM). The OMs were resuspended in a series of detergents in 50 mM Tris HCl, pH 8, 150 mM NaCl, 50 mM EDTA and 0.1 mg/ml 44 lysozyme. The OMs were suspended in different detergents: 2 % n-octylpolyoxyethylene (C8POE), 2% n-octyl-β-D-glucoside (OG) or 1% n-dodecyl-β-D-maltoside (DDM) and o homogenized with a Dounce homogenizer and kept stirring overnight at 4 C. The detergent o solubilized OMs were separated by ultracentrifugation at 100000 X g at 4 C for 60 min. Alternatively the cells were lysed by pressure homogenizer without spheroplast preparation. Unbroken cells were removed by centrifugation, and the supernatant was ultracentrifuged for o 100000 X g for 4 C for 60 min to separate the whole cell envelope. The whole cell envelope was suspended in cell resuspension buffer with DNase, and the suspension was carefully layered over discontinuous gradient made of 75%, 70%, 60%, 50% and 37% sucrose solution in HEPES buffer (pH 7.4). The step gradients were centrifuged in Beckman SW 32 rotor (80000 X g for 4 hr). The material that deposited at the 37% sucrose solution and above was collected, diluted with buffer 20 times and layered on 35% sucrose pad. The material was ultracentrifuged at 120000 X g for 3 hr. The supernatant was diluted 5 times with buffer and was pelleted by ultracentrifugation at 120000 X g for 2 hr. The pellet was stored as IM-enriched fractions. The material at the interface between 60 and 70% sucrose gradient was collected and stored as OM enriched fractions. For SDS PAGE, the protein samples were resuspended in 2X sample buffer (120 mM Tris HCl, pH 6.8, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol and 50 mg dithiothreitol). The samples were boiled for 5 min at 100o C, and the proteins were separated by 4-15% SDS-PAGE gel. Proteins were stained with Coomassie brilliant blue dye, and molecular weights were compared using a prestained protein marker ladder. 45 4.3.4 Mass spectroscopy The gel bands from SDS PAGE gels were cut and in-gel tryptic digestion was performed from the method modified form the published protocol. (141) Briefly, gel bands were dehydrated using 100% acetonitrile (ACN) and incubated with 10 mM dithiothreitol (DTT) and 50 mM iodoacetamide in 100 mM ammonium bicarbonate for 20 min each. Gel bands were then washed with ammonium bicarbonate and dehydrated again. Sequencing grade modified typsin was diluted to 0.01 µg/µL in 50 mM ammonium bicarbonate and ~50 µL of this solution was added to each gel band in order to completely submerge the gels. Bands were then incubated at 37o C overnight. Peptides were extracted from the gel by water bath sonication in a solution of 60% ACN/1% trichloroacetic acid (TCA) and vacuum dried to ~2µL. Peptides were then resuspended in 2% ACN/0.1% trifluoroacetic acid (TFA) to 20µL, and 10µL samples were injected by a Waters nanoAcquity Sample Manager TM and loaded for 5 min onto a Waters Symmetry C18 peptide trap and eluted to a Waters BEH C18 nanoAcquity column with a gradient of water and ACN. Eluted peptides were then sprayed into a ThermoFisher LTQ Linear Ion trap mass spectrometer equipped with a Michrom Bioresources Advance nano-spray source. Data-dependent zoom scans was performed onto the top five ions of each survey, followed by low energy collision induced dissociation, and the resulting MS/MS spectra were converted to peak lists using BioWorks Browser v 3.3.1 (ThermoFisher) with the default LTQ instrument parameters. All MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.4.0). Scaffold (version Scaffold 3.6.0, Proteome Software Inc., Portland, OR) was used to validate MS based protein and peptide identifications. 46 4.3.5 Biochemical characterization with UV visible spectroscopy Orientation of heterologously expressed OmcB to outer membrane was assayed by monitoring change in absorption spectra of oxidized versus reduced cytochromes by a UVvisible spectrophotometer. The reduction of low spin cytochrome was observed spectrophotometrically by presence of three characteristic absorption bands, called Soret or gamma peak which shift from ~408 in oxidized form to ~420 in reduced form, alpha (α) peak at ~ 552 nm and beta (β) at ~ 521 nm. Briefly 100 µL of cells at O.D. 2 were washed added to 900 µl of degassed resuspension buffer in a 1 cm path length quartz cuvette. The absorption spectrum of the cytochrome was measured by continuous scan from 380-600 nm. The cell suspensions were then reduced by addition of 5 µL of 0.5 % (w/v) freshly prepared sodium dithionite solution, and reduced spectrum was measured. The ability of cells to oxidize back by transferring electrons to an electron acceptor was tested by adding a soluble electron acceptor like Fe(III)nitrilotriacetic acid (FeNTA), ferric citrate, uranyl acetate (UA) riboflavin and or an insoluble electron acceptor like ferric oxide particles. Redox activity of membrane fractions was determined similarly using IM and OM preparations at 1-2 mg/ml concentration instead of cell suspensions, and reoxidation was achieved using FeNTA, ferric citrate, ferric oxide, or UA. 4.5 Results and discussion Fermentation conditions that gave maximum production of apoprotein were not suitable o to get maximum expression of mature protein. Cells grown at 30 C at 220 rpm without IPTG addition gave maximum yield of mature protein. Induction was not required due to 'leaky' expression of the plasmid. Addition of δ-aminolevulinic acid and hemin were essential for mature OmcB expression when grown with minimal media. The 2X YT medium without any 47 additional supplements was sufficient to express OmcB, but the amount of mature OmcB produced with 2 X YT media was lower compared to overall OmcB protein expression. o Lowering the temperature to 18o C after initial growth at 30 C slowed the OmcB production rates, but did not increase the ratio of mature protein expression. The MS analysis revealed the identified peptide cover 51% of the protein sequence (Fig. 4.1 ). As shown in Figure 4.1, 23 unique peptides were found, resulting 100% protein identification probability. The amino acid sequence was subjected to a BLAST search, which identified protein as cytochrome c protein from Geobacter sulfurreducens with 12 putative heme-binding sites. Subcellular fractionation of E. coli cells was used to localize heterologously expressed OmcB. The clarified cell expression media at the end fermentation, cell resuspension buffer after cell wash, and cytoplasmic fractions contained no measurable OmcB. An OmcB band was visible mostly in the membrane preparations and to a lesser extent in the periplasmic fraction. EDTA and lysozyme treatment breaks down bacterial outer membrane forming spheroplasts with intact inner membrane. (142) However, complete removal of OM from spheroplasts is challenging. (143) The sucrose density gradient method was used to separate to IM and OM fractions based on different buoyant densities of two membranes. However, cell lysis by pressure homogenization or ultrasonication may lead to formation of mixed membrane fractions. Hypertonic sucrose solution was used in the lysis buffer to minimize formation of mixed membrane pellets. (11) The gradient centrifugation method was optimized using various sucrose concentrations ranging from 20-70%. as well as different concentrations of divalent cations like MgCl2. As shown in Figure 4.3, the IM fractions were localized on the top of 37% the sucrose 48 gradient, while the OM fractions with higher densities were localized between 60 and 70% sucrose gradient. Both IM and OM fractions showed reddish coloration, but SDS PAGE revealed a very faint band at~ 80 kD for IM fractions, while the OM fractions showed a much darker OmcB band. Although sucrose density gradient was useful in separating E. coli membrane fractions based on density gradient, the IM and OM in E. coli are fused at several places (144), making the complete separation of the membranes difficult. N-lauroylsarcosine or sarkosyl detergent is commonly used to purify outer membrane proteins in E. coli and other bacteria (145147) because it efficiently dissolves cytoplasmic membranes while leaving outer membrane intact. Ultracentrifugation of membrane preparations after sarkosyl detergent treatment yielded dark reddish brown colored pellet of OM fractions. OmcB was a major protein present as a major band in OM preparations. The control OM preparations without OmcB plasmids were white in color and did not show any band in the 75-85 kD range. Yield of OM obtained using sarkosyl treatment was ~ 45 % of total membrane fraction. Compared to OM fractions, the supernatant of the ultracentrifugation step (referred as IM fraction) contain a very small amount of OmcB (Fig. 4.2). Similar results were obtained using 1 % Triton X-100 for differential detergent solubilization. Evidence of OmcB redox activity was obtained by monitoring UV visible spectra of E. coli cell suspension. The cell suspension buffer was purged with argon for 30 min, and the cuvettes used were flushed with nitrogen for 5 min. UV visible spectra of air oxidized cells showed a peak at ~ 410 nm, which shifted to 420 nm after reduction of cells with sodium dithionite. A small peak around 552 nm also became visible in the reduced cells. The reduced cytochrome was then oxidized with Fe NTA or ferric citrate, as evidenced by a Soret peak shift back to 410 nm and disappearance of the 552 peak (Fig. 4.3 A and B). A 520 nm peak often 49 associated with cytochrome c reduction (β peak), and a 530 nm peak with cytochrome c oxidation were not very apparent in these spectra, possibly due to higher scattering noise of the cell suspension. Similar reoxidation results were obtained when riboflavin and ferric oxide particles were used as oxidants (Fig.4.3 C and D). The reduction of ferric oxide, an insoluble electron acceptor that is unlikely to traverse through E. coli’s cell envelope, provides strong evidence that the expression host was able to package heme into the recombinant OmcB and target it to the outer membrane. A similar assay procedure was reported to analyze the orientation of outer membrane cytochromes in Shewanella oneidensis and heterologously produced OmcA in E. coli by reoxidation of reduced cells with soluble but cell membrane impermeable riboflavin. (148) Additionally substrate accessibility experiments with ferric NTA and insoluble iron oxide mediated reoxidation of sodium dithionite reduced cells concluded that type II secretion system was involved in the transport of heme domain to the outer surface of E. coli BL 21 strain. (47) Additionally those studies showed that heterologously expressed OmcA protein was released in the wash solution of the intact cells. In our experiments with OmcB expression we did not observe any release of OmcB in wash solution, which is consistent with the studies showing OmcB expressed in Geobacter sulfurreducens cells is only partially accessible to the outer membrane. (135) SDS PAGE analysis of subcellular fractions of the E. coli cells expressing OmcB showed the majority of OmcB to be membrane associated (Fig.4.2, and other data not shown). Inner and outer membrane fractionation by ultracentrifugation with sucrose density gradient and differential detergent extraction revealed majority of the heterologously expressed OmcB is associated with an outer membrane. UV visible analysis of outer membrane fragments showed that the recombinant OmcB was redox active toward a variety of substrates (Figure 4.5). Soluble 50 electron acceptors like FeNTA, ferric citrate, and UA reoxidize sodium dithionite reduced OmcB OM fractions. Insoluble electron acceptors like iron oxide particles were also able to interact with OmcB OM fractions to reoxidize it back. Interestingly another outer membrane cytochrome of Geobacter, OmcZ, was not redox active towards insoluble electron acceptors like iron oxide. (138) This finding is consistent with the observation that OmcZ is released into extracellular matrix while OmcB remains associated with the outer membrane of Geobacter and may be important for localized reduction of metals at the bacterial surface. Artifacts generated during membrane protein isolation protocols can lead to formation of mixed membrane fragments (IM and OM); also presence of insoluble periplasmic aggregates can be associated with OM during cell fractionation, leading to false conclusion about the localization of overexpressed protein in E. coli. (149) Fractionation with two independent techniques (sucrose density gradient separation and differential detergent extraction), along with whole cell spectroscopy to believe that heterologously expressed OmcB is indeed oriented in the outer membrane of E. coli. The membrane association of OmcB was found to be very strong and resistant to a variety of detergent treatments. A cysteine mutation with alanine in the OmcB leader sequence yielded detergent extractable protein (OmcBc1a). However, UV-visible spectroscopy suggested that the redox activity of this mutant protein did not match the expected cytochrome c profile. The Soret peak of the cysteine mutant shifted to 428 nm, with additional peaks at 506, 540 and 560 nm (Fig. 4.6). Therefore it may be possible that the cysteine residue in the protein sequence is important for the redox activity of the protein. The genome of Geobacter predicts around 100 membrane associated cytochrome. (57) The high number of cytochromes involved in the electron transport in this bacteria, makes the study of individual cytochromes extremely challenging. Because of high number of cytochrome 51 present in these bacteria, there seem to be a functional redundancy. Even though the knock out models can be used to study contribution of specific cytochromes, the bacteria tend to adapt over the time by expressing other cytochromes. One strategy is to use heterologous expression of these Geobacter proteins in other hosts like E. coli. The heterologous expression can be either used to build a synthetic chain of redox active proteins in bacteria (150) or to purify the protein and study its biochemical properties in detail. (151) However the heterologous expression of these Geobacter outer membrane protein in other hosts in challenging, because: 1. The protein needs to be transported across the inner membrane of the bacteria by a pathway like Sec pathway. (152) 2. The apoprotein need to be matured in the periplasmic space with the help of heme maturation machinery in the bacteria, which involves several different proteins such as cytochrome c maturation proteins a, b, c, d, e, f, g , h, and I. (53) 3. The protein then need to be transported to the outer membrane, which also requires specific mechanism like Type II secretion system. (44) Due to these challenges, very few multiheme cytochromes from Geobacter have been expressed in E. coli.(85, 153) A high-throughput scheme has been proposed to express the multiheme cytochromes in E. coli with novel expression vectors. (154) But this scheme utilizes only soluble part of the protein to facilitate the purification. The proteins expressed with such system may not aid in the cellular localization and functional characterization of these proteins. To the best of our knowledge, we are the first group to express multiheme, outer membrane cytochrome from Geobacter in E. coli. The advantages of using the whole sequence of the OmcB protein as opposed to only soluble part for the heterologous expression include that 52 the expressed protein is more likely to be in similar in characteristics to the native OmcB. Another advantage of using E. coli host is that under aerobic conditions E. coli does not produce cytochromes maturation proteins. The OM fractions separated from control cells of E. coli transformed with only pEC86 plasmid, did not show any UV-visible signature peaks for cytochrome c. Lack of native cytochromes in E. coli is of advantage to us because it means that any cytochrome signal obtained from E. coli membrane must be from the heterologously expressed cytochrome. This results enables us to use the membrane vesicles of E. coli for further studies of redox activity of OmcB. This saves the efforts of purifying the protein to isolation before studying its redox properties, but more importantly we can study the protein in the environment which is more likely to preserve its activity. Since OmcB sequence indicates putative membrane spanning domains (155) and other studies indicate that OmcB may be embedded in the outer membrane with only partially exposed to the solution side (156), keeping the protein associated with the membrane is more likely to preserve its function. We aim to study this protein further using a biomimetic interface, where the membrane vesicles form E. coli containing OmcB will be incorporated to study its electrochemical and spectro-electrochemical properties. In summary, we demonstrated that heterologously expressed OmcB in E. coli cells is membrane associated with majority of protein present in the outer membrane. The OmcB is accessible to and active with a variety of redox partners. These results are a key step towards further analysis of this important redox protein in understanding the redox chemistry of Geobacter in vitro. 53 5.0 SPECTROELECTROCHEMICAL STUDIES OF GEOBACTER REDOX PROTEINS EXPRESSED IN E. COLI 5.1 Introduction Spectroelectrochemical technique is a powerful analytical tool offering advantage of both electrochemistry and spectroscopy, allowing quantitative and qualitative characterization of an analyte as a function of changes in electrode potential. A variety of spectroscopic techniques can be coupled with electrochemical analysis which can follow changes in structure of analyte with its redox activity. Examples of the spectroscopic techniques that can be combined with electrochemistry include but are not limited to are electron paramagnetic resonance (EPR) or electron spin resonance which can be useful to study paramagnetic systems such as transition metals and radicals (163-165), vibrational spectroscopies such as Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy (166-170), and UV- visible spectroscopy. (171174) UV-visible analysis coupled with electrochemistry has been used to study the redox reactions of biomolecules. (175-177) Information about the redox thermodynamics of the proteins (178) and kinetics of their reduction reactions with substrates (179) can be obtained by spectroelectrochemical studies. Developments in the optically transparent electrode materials have made it possible to study a variety of biological samples directly on solid substrate. Protein samples can be analyzed by adding them to the protein/lipid films (180, 181), supported lipid bilayers (182), by depositing partially dehydrated protein films on the surface (183) or by immobilizing on an optically transparent electrode. (184, 185) UV-visible spectra of cytochromes can be easily distinguished because of the heme groups associated with the protein. By monitoring changes in the UVabsorption of heme groups because of Π - Π * transition (167), the information about the type 54 of heme, its oxidation state and spin state of iron in the porphyrin ring can be obtained. (167) Further information about the resolution of individual hemes in multiheme cytochrome (186) and conformational changes in individual hemes in different redox states (186) can be obtained by these techniques. Design of optically transparent cell geometry and selection of electrode material are crucial to build an optimal spectroelectrochemical system. The ideal electrode material should have minimum ohmic resistance, maximum surface area to volume ratio, and wide spectral and potential window. (187) Deposition of optically transparent thin conductive films made of metals (188), metal oxides (189), and carbon material (190, 191) on the substrate offers a variety of spectral regions to be studied. Optically transparent thin cell electrode (OTTLE) system offers several advantages which include (187, 192) 1. Complete and rapid electrolysis of the redox sample. 2. Possibility of using a variety of electrode material like boron doped diamond or SAM coated gold electrode. 3. Requires a small amount of samples, which can be very useful when working with recombinant proteins. 4. Possibility of taking measurements in anaerobic conditions, which can be helpful when working with oxygen sensitive proteins or substrates. 5. Quantitative relationship between experimental and theoretical spectroelectrochemical parameters. In this study, we have made efforts to develop a substrate on the transparent gold electrodes which will protect the protein in solution from degradation at the electrode while allowing rapid electron transfer. The substrate developed also offers a possibility of 55 immobilizing the protein on the electrode in an electroactive configuration. We have also explored the possibility of using OTTLE for spectroelectrochemical studies of Geobacter multiheme cytochromes heterologously expressed in E. coli. We have demonstrated that PpcA is capable of direct electron transfer to metal ions when immobilized on a SAM coated gold electrode. Furthermore we have also studied the electrochemistry of outer membrane protein OmcB embedded in membrane vesicles with different redox mediators. Moreover, we have demonstrated that periplasmic protein PpcA is capable of electron transfer to OmcB. To the best of our knowledge, we are the first group to show evidence of protein-protein electron transfer in OTTLE. 5.2 Materials and methods 5.2.1 Thin-layer spectroelectrochemical system Schematics of the OTTLE cell are shown in Figure 5.1. (187) The OTTLE cell consists 2 of a holder for the working electrode, which can be 2cm X 2cm with 0.78 cm working area. The working electrode (WE) is pressed against the sample holder with a transparent window made of calcium fluoride (CaF2) and is the WE separated from the window by a thin spacer. The thickness of spacer can be varied from 25 µM to 125 µM, and thus the volume of the OTTLE cell can be controlled. The diameter of the spacer is slightly larger (1 cm) than the diameter of the transparent window (0.9 cm). An O-ring seals the volume of the spacer. A small fine Pt wire connected to the holder serves as a counter electrode. A small amount of electrolyte such as KCl is added to the counter electrode capillary compartment to complete the circuit. A miniature Ag/AgCl reference electrode is also connected to the counter and working electrode through a fritted glass. 56 5.2.2 PpcA and OmcB cytochrome c The protein samples of PpcA and OmcB were prepared using the methods described in chapter 2 and chapter 4 respectively. PpcA samples were dissolved in degassed phosphate buffer (50 mM, 150 mM NaCl, pH 7.2), and the concentration was adjusted from 700 µM to 10 µM. OmcB membrane vesicles were stabilized by detergent (0.5% n-dodecyl, b-D-maltoside (DDM)) and sucrose (100-500 mM). OmcB content of the total protein in membrane vesicles determined by densitometry of SDS PAGE gels was from 40-70%. 5.2.3 Electrochemical and Spectroscopic measurements UV-vis measurements were made by placing the spectroelectrochemical cell in the path length of a Hewlett Packard diode array spectrophotometer (model 8453 Agilent Technologies, Santa Clara, CA). At the beginning of each experiment the optical and electrochemical measurements were synchronized. The electrochemical experiments were performed using CH instrument potentiostat (model 660 b, 760 b, and model 830 c; CH instruments Inc, Austin TX). The potential values reported here are against Ag/AgCl reference electrode. The cathodic current values reported here are positive, and anodic current are reported negative. The data files were saved in ASC II format and were imported into MS excel format™ or plotted by Igor Pro™ software. Baseline noise reduction is achieved by subtracting the average of spectra before the oxidation / reduction step from the following spectra. Electrochemical techniques used included cyclic voltammetry (CV), chronoamperometry (CA) and multipotential step voltammetry. A more detail explanation of these techniques is give in previous chapters. For multipotential step voltammetry, the potential of the working electrode is changed in small increments and kept constant at each potential for specified time. The change 57 in current is noted as a function of change in potential. Addition of all the samples to OTTLE were done inside a glove box in nitrogen environment. All the samples and buffers were thoroughly degassed using argon. Spectroelectrochemical reduction of OmcB membrane vesicles with a mixture of mediators was characterized. The mediator stock solution contained 1 mM of each of the following freshly made mediators: gallocyanine, phenazine methosulfate, 2-hydroxy1-4 naphthoquinone, anthraquinone 2 sulfonic acid, benzyl viologen, and methyl viologen. The mediators were chosen to cover the potential range of interest (+21 mV to -430 mV against SHE). The concentration of mediators used was around 50 µM. The spectra of mediators were recorded using multipotential step voltammetry. Then the spectrum of OmcB was recorded, with and without the mediator mixture. 5.2.4 Preparation of working electrode o The working electrode was a gold coated glass cover slide with 100 A thickness of gold coating (Sigma-Aldrich) or the custom made electrode with gold grid printed on quartz. The working electrodes were cleaned by Piranha solution or oxygen plasma cleaner. The cleaned electrodes were immediately contacted with ethanolic solution of alkanethiols (~ 5 mM concentration). The chain length of alkanethiols were varied from 6-16 C, and the ratio of carboxy terminal to hydroxy terminal group was adjusted. Protein solutions were contacted either directly with working electrode (for PpcA) or with the help of redox mediators (for OmcB). PpcA was then immobilized on the SAM covered gold working electrode by the procedure described in chapter 2. 58 5.3 Results and Discussion 5.3.1 Immobilization of PpcA on optically transparent gold electrodes. The negatively charged SAM deposited on gold surface electrostatically attracts the positively charged PpcA protein at neutral pH. No current was obtained when PpcA protein was initially immobilized using C-16 SAM containing 16-mercaptohexadecanoic acid. However, the number of carbons in the lipid and the ratio of hydroxyl to carboxyl terminated groups in the SAMs were adjusted to optimize the faradic current. We chose mixture of 12mercaptododecanoic acid with 11-mercapto1-undecanol or 6-mercaptohexanol in 1:3 molar ratio for immobilizing the cytochrome on the electrode. The immobilized PpcA displayed distinct redox peaks for oxidized and reduced forms. The ratio of peak current to the scan rate was linear, consistent with the electrochemistry of the adsorbed protein. The coverage of active adsorbed 2 protein, obtained from integrating the area under CV peak is calculated to be 18 pmol/cm . The peak half width (the difference in potential at half the maximum current value) was ~ 140 mV, much higher than the expected value for the reversible species (84), but the redox peaks were stable during multiple runs (data not shown). This finding leads us to believe that the protein was getting completely oxidized and reduced at the electrode reversibly, but maybe due to a large number of lysine residues available. The positively charged lysine residues are attracted towards negatively charged SAM, but because there are multiple residues, the adsorbed protein might be in a range of orientations. Stability of the adsorbed protein was monitored by square wave voltammetry by following decay in the signal. The square wave voltammograms were recorded with amplitude of 50 mV, frequency of 25 Hz, and potential step of 2 mV. The adsorbed cytochrome c was very stable for at least 6 hr and the peak current reduced to ~80% of its value after 24 hr (Fig. 5.2). Furthermore CV were recorded at the fast scan rates, and the data obtained 59 were fitted to the Laviron equation (please refer to chapter 2 for more details) to find the heterogeneous electron transfer rate constant between PpcA and electrode. (data of this section is not shown) Interestingly, we did not observe any peaks associated with cytochrome c during the UV-vis spectroscopic analysis of the optically transparent gold substrates with immobilized PpcA. This observation is not surprising, because of the small size of the protein (~ 2 nm) at the monolayer coverage will not have enough protein at immobilized surface to give UV-vis spectra. Another group working with immobilized cytochrome on a similar SAM reported UV spectra for the protein adsorbed at the monolayer, with the possible explanation that surface plasmons associated with the gold electrode enhance the signal of protein. (184) However, we did not notice such signal augmentation in our studies. Nonetheless, the protein immobilized at the monolayer at the optically transparent electrode provides a possibility of monitoring a redox reaction of proteins with substrates, where the changes in UV-vis spectra of the substrate can be monitored as a function of redox reaction mediated by an immobilized cytochrome. 5.3.2 Spectroelectrochemical characterization of PpcA in solution Electrode fouling due to adsorption of the inactivated protein (193) or denaturation of proteins (194) during electrochemical measurements at the bare gold substrate can pose experimental problems. Therefore the PpcA was characterized spectroelectrochemicaly with the C-6 SAM coated gold. Distinct oxidation and reduction peaks were observed with 10 µM solution of PpcA on SAM coated gold electrode. The peak potential separation increased with increased scan rates (Fig. 5.3), and the peak current value increased linearly with square root of scan rate as expected for the reaction controlled by the diffusion of the species (Fig. 5.3 inset). Spectroscopic changes associated with the reduction of PpcA at SAM coated electrodes are shown in Figure 5.4A. PpcA cytochrome is capable of direct electron transfer with the electrode 60 without any redox mediators. Therefore the spectroscopic signal of PpcA in solution is dominated mostly by changes associated with the redox center of the protein of interest and not of any mediators. Figure 5.4 B shows changes in absorption spectra of PpcA at the OTTLE for various electrode potential values. The wavelength values characteristic of the cytochrome c protein (315 nm, 408 nm, 420 nm, 520 nm, 530 nm and 552 nm) display the most noticeable changes in absorbance. Moreover the differential spectra also show changes at 280 nm and 430 nm range, which may be associated with the changes in the peptide arrangement of the protein in different redox states. Redox performance of the PpcA solution was monitored by CA measurements, where the potential of the electrode was changed to a large overpotential value (in this case reduction potential of -600 mV vs. Ag/AgCl) and the resulting change in the current was measured. The Cottrell equation (84) describes the relationship between current value (i) at the planar electrode with diffusion coefficient and time as follows: 𝑖= 1� 2 𝑛 𝐹 𝐶𝑜 𝐷𝑜 1� 1� 2𝑡 2 Π .............................(Eq. 5.1) where, n is the number of electrons transferred in the reaction, F is the Faraday's constant, A is the area of the electrode, and Co and Do are the concentration and diffusion coefficient, respectively, of the electroactive species. Chronoamperometry experiments were repeated at the multiple potential steps, and the change in current were monitored. The change in current following a potential step for PpcA (Fig 5.5 A) shows a behavior typical of the diffusion species. Interestingly, the change in the absorbance at 552 nm also shows similar behavior, though at apparently slower rate, possibly because the CA measurements follow the change in current mostly at the electrode surface, while the spectroscopy data follow the change in the whole volume of the cell. A common trend is that both signals reach a plateau at around 300 sec, 61 indicating almost complete reduction of PpcA in solution at that time point. Figure 5.5 B shows the reversibility of the PpcA absorbance signal during multiple CA run, indicating that the SAM of alkanethiols protects the cytochrome from degrading and the electrode from fouling. 5.3.3 Spectroelectrochemical characterization of OmcB in OTTLE OmcB membrane vesicles were not found to react with the electrode directly, as evidenced by no change in current with a change in applied potential during CA and CV measurements. However, with the addition of mediators, Soret peak absorbance values at 420 and 552 nm peaks were observed that suggested OmcB redox activity (Fig. 5.6). The changes in absorption spectra with changes in electrode potential were analyzed by the modified Nernst equation (195) : 𝐸 = 𝐸 𝑜′ + 𝑅𝑇 𝑛𝐹 𝑙𝑛 𝐴−𝐴𝑟 𝐴0 −𝐴 ............................(Eq. 5.2) o' where, E is the potential of the electrode, E is the formal potential of the redox couple, A is the absorbance, Ar and Ao are the absorbance of the fully reduced and fully oxidized species. R, T, n and F are ideal gas constant, absolute temperature, number of electrons transferred, and Faraday's constant respectively. For the electrochemical reaction of proteins in presence of mediators, the Nernst equation can be rearranged in the following form (195, 196) : 𝑜′ 𝐸 = 𝐸𝑀 + 𝑅𝑇 𝑛𝐹 [𝑂 ] 𝑙𝑛 [𝑅𝑀] = 𝐸𝑃𝑜′ + 𝑀 𝑅𝑇 𝑛𝐹 62 𝑙𝑛 [𝑂𝑃 ] �𝑅𝑝 � ..............................(Eq. 5.3) o' o' Where, E M and E P are the formal potential of the mediator and protein respectively and OM, RM, OP and RP are the concentration of oxidized mediator, reduced mediator, oxidized protein and reduced protein respectively. From plotting the data of percentage of OmcB reduced (from the change in 410 nm peak) vs the potential of the electrode, and fitting the curve to Nernst equation, a tentative midpoint potential value for OmcB was obtained. Fig 5.7. Furthermore, by carefully changing the electrode parameters it may be possible to further resolve the individual hemes or at least groups of hemes using this technique. (197) Fig 5.7 shows possible resolution of individual heme groups as a the potential was varied. Moreover we found a tentative range for the redox potential of OmcB from these experiments (~ -400 mV vs. Ag/AgCl). In another set of experiments, the mediators were replaced with 50 µM solution of anthraquinone 2,6-disulphonate (AQDS) which serves as a humic acid analogue. (198) OmcB was reduced and oxidized reversibly figure 5.8), indicating that AQDS serves as a redox mediator for OmcB. Quinone moieties are reported to facilitate the extracellular electron transport in Geobacter and enhance the rates of bioremediation processes. (199) Considering the outer membrane localization of OmcB, it may be possible that OmcB is one of the c cytochrome involved in electron transfer to humic acid moieties found in soil and sediments. 5.3.4 Spectroelectrochemical characterization of PpcA -OmcB interactions Based on the hypothesis that PpcA may be transfer electrons produced in respiration to OmcB as part of Geobacter’s electron transport chain, we attempted to demonstrate electron transfer from PpcA to OmcB in OTTLE cell. The CV of PpcA at the scan rate of 50 mV per s shows a distinct redox peak. Addition of OmcB-containing membrane vesicles to the PpcA, 63 yielded a small increase in the CV peak (Fig.5.9). Since OmcB membrane vesicles, are not capable of direct electron transfer to and from the electrode, the increase in the current can be attributed to the catalytic reduction of OmcB by reduced PpcA. Redox spectra of the cytochromes monitored in the multipotential step experiments showed additional evidence of reduction of OmcB from PpcA (Fig. 5.10 A). Change were observed in the Soret Peak at 410 and 420 nm, and 552 peaks as a function of electrode potential. In the case where only PpcA is getting oxidized and reduced at the electrode, the 410 peak associated with oxidized OmcB will not change. Thus at the reducing potential of PpcA a mixture of peaks in the Soret regions would be observed. The complete disappearance of the 410 peak at reducing potential and complete removal of 420 peak at the oxidizing potential provides the evidence that both cytochromes are getting oxidized and reduced. Spectra of OmcB in multipotential voltammetry can be obtained by subtracting the spectra of PpcA from the spectra of both the cytochromes together (Fig. 5.10 B). Since OmcB was not observed to interact with the electrode, PpcA is apparently acting as a redox mediator between electrode and OmcB. The biomimetic system assembled here was intended to simulate electron transfer within Geobacter’s cell envelope, in which electrons produced in respiration are transferred from the inner membrane (mimicked by the SAM coated gold electrode) through the periplasm (mimicked by the PpcA layer bound to the SAM) to outer membrane cytochromes (mimicked by the OmcBcontaining microsomes. This study’s establishment of a biomimetic redox-active cell envelope (BRACE) provides a new research platform with which to study Geobacter electron transport to extracellular electron acceptors. The use of PpcA and OmcB heterogeneously expressed in E. coli helps eliminate the possibility that other Geobacter cytochromes that might be difficult to separate from the PpcA 64 and/or OmcB are participating in the electron transfer. Assuming the redox properties of the heterogeneously expressed OmcB are similar to those of the native OmcB, these experiments support the hypothesis that periplasmic cytochrome PpcA is able to transfer electrons to the outer membrane cytochrome OmcB. Thus OTTLE system provides two independent measures of redox activity (optical and electrochemical) that provide more insight into the cytochrome electron transfer than either method alone. The ability to simultaneously interrogate a BRACE both optically and electrochemically offers the potential to significantly accelerate progress in elucidating mechanisms of Geobacter electron transfer and testing hypotheses about system behavior. For example, another soluble cytochrome could be substituted for PpcA, and the resulting changes in spectroelectrochemical characteristics could be observed. Similarly, vesicles containing different outer membrane cytochromes could be used, and the relative efficiency of inter-cytochrome electron exchange between PpcA and the various outer membrane cytochromes could be explored. This approach could provide insight into the principal redox pathways Geobacter uses to transport electron transport to metals. 65 6.0 ENGINEERING A BIOMIMETIC REDOX ACTIVE CELL ENVELOPE WITH HETEROLOGOUSLY EXPRESSED GEOBACTER CYTOCHROMES 6.1 Introduction Biological systems are often complex in nature, with heterogeneous biomolecules arranged in a specific spatial hierarchy. (200) Such spatial arrangement plays a crucial role in controlling protein-protein interaction, for example close proximity of the proteins reduces the time required for diffusing protein to find the target protein. (201) Metabolic processes take place where multiple species are working in tandem, localized in specific cell compartments. Synthetic biologists and metabolic engineers have been working to understand the multidimensional aspect of these biological processes, yet characterizing these pathways remains challenging. (202) There is an ever increasing demand about understanding these biological routes, in order to optimize the commercially and environmentally important bioprocesses. Dissimilatory metal reduction mediated by anaerobic bacteria Geobacter is an example of such important process because of its application in uranium bioremediation and microbial fuel cell technological applications. (203, 204) Geobacter display a large number of electron carriers which are located in different cell compartments (57) for e.g.: inner membrane (e.g. cytochrome bc complex), periplasmic space (e.g. PpcA cytochrome), outer membrane (e.g. OmcB), and the pili protein protruding out of the membrane. In this work we try to address some key issues about the Geobacter electron transfer (ET) process: 1) Is there a possible interaction between the two of the most well conserved Geobacter cytochromes (56) (PpcA and OmcB), and 2) Are there any tools available to study these complex and compartmentalized processes in a more controlled manner. Our strategy is to build a nanoscale biomimetic system where these proteins can be 66 studied in a close proximity yet separated by different compartments similar to the cell envelope of the native bacterium. Artificial lipid bilayers have emerged as a very useful tool to mimic structure of biological cell membranes. (205, 206) Use of biomimetic membranes allows to study characteristics of biological moieties in an environment which gives more control over experimental parameters without sacrificing functional properties of biomolecules. (207-209) Traditional methods like “patch clamp” to study biological membrane have long given a way to more sophisticated techniques like planar bilipid membrane, in which lipid bilayer is painted over small aperture separating two electrolyte chambers. (210-212) Although this is very sensitive method, the bilayer formed by this method is very fragile. (212) More robust artificial bilayer can be formed by supported bilayer lipid membranes (sBLMs). (213, 214) sBLMs can be deposited on a variety of substrates including surface like gold, (215, 216) glass, (217) silica, (218) and mica. (219) Deposition of lipids by vesicle fusion around the protein immobilized on a glass surface showed that the lipid still maintained its fluidity. (220) Incorporation of membrane proteins into sBLMs using proteoliposomes facilitates study of their structural and functional properties. (221) (222) We have adopted similar methodology to built our proposed biomimetic interface. In this study we have deposited proteoliposomes on a self assembled monolayer (SAM) containing immobilized protein. Rupture of proteoliposomes vesicles on the SAM produces highly insulating film on surface. Because of this blocking layer, any electron transport (ET) must takes place via membrane proteins immobilized in the upper lipid layer of the interface. In this chapter we have demonstrated that the biomimetic redox active cell envelope (BRACE) allows us to study ET in a biomimetic system containing multiple cytochromes. Although more work about BRACE protocol optimization and studies to establish its architecture 67 needs to be done, we have demonstrated with the use of BRACE that ET is feasible between periplasmic cytochrome PpcA and outer membrane cytochrome OmcB, making them possible redox partners. We are the first group to report use of BRACE to study ET between different Geobacter cytochromes and these results suggest that BRACE interface can be used to study the ET between different redox proteins in a more controlled environment. 6.2 Materials and methods 6.2.1 Preparation of protein samples Please refer to chapter 2 for a detail description about heterologous expression and purification of Geobacter periplasmic cytochrome c protein: PpcA. Similar method of expression -heme was used to produce PpcA except in this case the E. coli cells were transformed with only PpcA plasmid. Omission of pEC86 plasmid necessary for the heme incorporation resulted in -heme expression of apocytochrome without heme. PpcA was purified from periplasmic fraction using cation exchange chromatography and the samples were collected following UV signal. The samples were analyzed by SDS PAGE to verify the presence of the protein. Unlike PpcA, the -heme PpcA is a colorless protein and does not show any UV peaks associated with cytochrome c (at 410, 420, and 552 nm on reduction). The PpcA protein samples were stored in phosphate buffer pH 7.2, 100 mM NaCl. Before immobilization experiments, the protein samples were either buffer exchanged or diluted to salt concentration of around 10 mM. Please refer to chapter 4 for description about isolation of outer membrane vesicles from E. coli, expressing Geobacter outer membrane cytochrome OmcB. Outer membrane fractions form OmcB with cysteine mutation (OmcBc1) were also isolated with the similar procedure. 68 The outer membrane vesicles were suspended in 10 mM HEPES buffer containing 150 mM NaCl and sonicated with 20 s pulse and cooling for 40 s between each pulse until the suspension become clear (~10 min). The suspension was then filtered through 450 nm filter. 6.2.2 Preparation of DOPC liposomes The phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Avanti Polar Lipids Inc., Alabaster, AL. A chloroform solution of DOPC required to make 1 mM lipid was dried under the stream of nitrogen until it forms a thin white lipid cake. Then the lipids were freeze dried for at least 4 hr. The dried lipids were rehydrated with HEPES buffer (10 mM HEPES, 150 mM NaCl, 2 mM CaCl2, pH 7.4) to form multilamellar vesicles, which were ultrasonicated in bath sonicator for 30 min to obtain small unilamellar vesicles. For formation of proteoliposomes, OmcB outer membrane vesicles were added before or after the sonication of DOPC lipids. In an alternate protocol, the DOPC liposomes were contacted with 0.5 % n-dodecyl-β-D-maltoside (DDM) detergent stabilized OmcB vesicles and dialyzed overnight with HEPES buffer using a 30 kD molecular weight cutoff membrane. 6.2.3 Fabrication of compartmentalized bilipid membrane on gold electrode Commercial gold substrates (LG thin films, Santa Clara, CA) were cleaned by dipping in Piranha solution (7 parts sulfuric acid and 3 parts 30% hydrogen peroxide solution) for 30 s and then rinsing with distilled water. For SAM formation, the cleaned substrates were dipped in 5 mM ethanolic solution of 12-mercaptododecanoic acid and 11-mercapto 1 undecanol in a 1:3 ratio for 24 h, and then rinsed with ethanol and distilled water. PpcA was bound to the SAM via an amide linkage using 50 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 5 mM N-hydroxysuccinimide (NHS) solutions. The electrodes were then washed in 20 mM phosphate buffer to remove unbound protein. DOPC liposomes or the proteoliposomes 69 were added to the immobilized PpcA substrate to the final concentration of 500 µM of lipids. The substrates were kept overnight to form the bilayer on the surface, after which the solution was replaced with phosphate buffer, pH 7.2, 100 mM NaCl. Gramicidin activity was tested by adding 1 µM of protein from ethanolic stock solution. 6.2.4 Analytical techniques The mean diameter proteoliposomes and particle size distributions of DOPC liposomes or were determined by dynamic light scattering (DLS) using a 90 plus nanoparticle size analyzer (Brookhaven Instruments Inc, NY). Liposome solutions were diluted to an appropriate dilution with buffer and measurement were obtained at 90o. Twenty size determination runs were performed for each sample. A 15 µL aliquots of samples were deposited on a Formvar grid and stained with 2% uranyl acetate. Transmission electron microscopy (TEM) Images were acquired using JEOL 100CX transmission electron microscope (JEOL USA Inc., NJ). Microgravimetric analysis was done using a quartz crystal microbalance (QCM) (Research Quartz Crystal Microbalance, Maxtek Inc., Santa Fe Springs, CA) controlled by Maxtek RQCM Data Log software. The piezoelectric quartz crystals with 5 MHz oscillating frequency, was held between two gold electrodes. The QCM electrodes were first cleaned by rinsing with ethanol and drying under the stream of nitrogen. Then the crystals were cleaned using an oxygen plasma cleaner. The cleaned crystals were immediately contacted with ethanolic solution of 5 mM mercaptolipids to form SAM. After SAM formation, the crystal were also used to immobilize PpcA. The crystals ready to analyze were mounted on a crystal holder and changes in frequency changes were monitored after addition of DOPC liposomes. 70 Ellipsometric measurements were carried out using M-44 rotating analyzer ellipsometer ( J.A. Woollan Co. Inc., Lincoln, NE) operated by WVASE32 software. The refractive index and the extinction coefficient values were assumed to be 1.5 and 0 for all measurements. The bilayers were removed from the solution just before the measurements. For DOPC bilayers measurements, the film was modeled as a transparent “Cauchy” layer with refractive index of 1.5. Electrochemical analysis was performed using CHI 660 B and CHI 660 D potentiostat (CH Instruments Inc, Austin, TX). Electrochemical techniques used were cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). For more details about the principles of CV, please refer to earlier chapter. EIS is a nondestructive electrochemical technique, which measures the impedance (the net resistance to current flow offered by the interface) at an applied potential. EIS studies were carried out at a bias DC potential (often a open circuit potential) using small sinusoidal potential within a frequency range of 0.01 to 10,000 Hz. The impedance data for the interface was fitted to a Randles equivalent circuit model which include electrical elements such as capacitors and resistors. (84) The impedance (Z) of the system is represented by: 𝑍 (𝜔 ) = 1 𝑌(𝜔) 𝑉 = �cos(𝜃) − 𝑖 𝑠𝑖𝑛(𝜃)� = 𝑍𝑟 + 𝑖 𝑍𝑖 ......................(Eq. 6.1) 𝐼 where, Y is the admittance, V is the voltage, I is the current, θ is the phase delay, and ω is the radial frequency. Zr and Zi are the real and imaginary parts of an impedance. The impedance data is plotted as real vs. imaginary part of the impedance (Nyquist plot); or as the plot of absolute value of impedance vs. frequency function (Bode plot). Addition of films on the surface 71 of electrode increases the charge transfer resistance (Rm) of the electrode. With addition of each successive layer, the Rm value increases, which help monitoring the interface formation. The impedance data was analyzed using Z -view software (Scribber Associates, NC). 6.3 Results and discussion 6.3.1 Characterization of DOPC liposomes and microsomes The TEM data show the formation of DOPC liposomes with particle size of about 100 nm in diameter (Fig 6.1 A). The DLS analysis confirmed the size with mean diameter of DOPC liposomes of 110 ± 4 nm. The proteoliposomes measured by DLS had mean particle size of 170 ± 8 nm. The proteoliposomes showed a tendency to aggregate within hours of formation, therefore only freshly formed proteoliposomes were used to form BRACE. 6.3.2 Characterization of BRACE using QCM QCM is a routinely used technique to monitor deposition at the solution surface interface. (223, 224) Fusion of DOPC liposomes and its kinetics of its rupture on SAM coated gold electrode to form a bilayer was monitored by QCM . In this method we measured changes in oscillating frequency (∆f) of the crystal due to adsorption of the liposomes. The correlation between ∆f and mass of the adsorbed species is give by Sauerbrey equation (225, 226) : Δ𝑓 = −2Δ𝑚𝑓2 𝐴��𝜇𝜚𝑞 � = −𝐶𝑓 Δ𝑚 .........................Eq. 6.2 Where, ∆m is the change in the mass, f is the intrinsic frequency of the crystal, µ is the shear modulus, A is the electrode area, and 𝜚𝑞 is the density of the quartz. 72 Figure 6.1 B show plot of ∆f over the time after addition of liposomes to the SAM coated gold electrode. The curve display a rapid decrease in frequency followed by a slow increase to a steady-state value of lower frequency. This behavior is characteristic of two phase liposome interaction on the surface. (227) In initial phase, the rapid decrease is due to the adsorption of intact liposomes on the surface of the crystals, resulting in decrease in oscillating frequency. In second phase, the liposomes rupture on the surface to form a bilayer. The resultant release of entrapped water in the liposomes reduces the mass of adsorbed species, resulting in slight increase in the frequency. This trend is consistent with the hypothesis, that liposomes rupture on the SAM surface to form a bilayer, though we cannot completely rule of attachment of some non ruptured liposomes on the surface. Ellipsometric characterization of formed after DOPC addition o revealed the deposition of film ~75 A in thickness, which may also indicate formation of lipid bilayer on the surface. 6.3.3 Electrochemical characterization of BRACE A representative EIS data of BRACE formed with OmcB proteoliposomes is shown in Figure 6.2A. Formation of BRACE was analyzed by fitting the EIS data to the Randle's modified equivalent circuit model as shown in Figure 6.2B. In this model, the double layer capacitance was replaced with constant phase element to accommodate the hydrophilic and heterogeneous lower portion of BRACE. (228, 229) Z value software was used to calculate the membrane 2 resistance (Rm) values of 1.2 MΩcm 2 and 1.38 MΩcm 2 for liposomes and proteoliposomes. 2 The capacitance values were 0.51 µF/cm and 0.59 µF/cm for BRACE formed with liposomes and proteoliposomes. In both cases the data represents formation of a highly insulating lipid layer. The membrane resistance increased steadily till 6 hrs and reached a steady state. Therefore 73 the membranes prepared in all the subsequent studies were kept overnight for formation of stable and insulating BRACE. Periplasmic cytochrome PpcA, immobilized on a SAM display a redox peak in CV measurements. Addition of ferric nitrilotriacetate (FeNTA) solution to the interface results in increase in the catalytic current of the protein (data not shown). Addition of DOPC bilayer to form a BRACE, does not block the redox interaction of PpcA with the underlying gold electrode. However, addition of Fe NTA solution to the BRACE made with DOPC liposomes does not result in increase of the catalytic current (Fig. 6.3A). This is consistent with the formation of the highly insulating lipid bilayer blocking the access of electron acceptors in solution forming separate compartments on the electrode. Formation of BRACE with proteoliposomes containing OmcB protein did not result in observable change in CV signal of PpcA. This may be due to lower amount of membrane protein OmcB getting immobilized in the vicinity of PpcA. This is consistent with observation that immobilized PpcA do not show observable increase in the catalytic current with soluble electron acceptor like Fe NTA until a threshold level is reached (~200 µM). The concentration of OmcB incorporated in proteoliposomes is much smaller (~ 10 µM). However, the BRACE formed with OmcB proteoliposomes displayed noticeable increase in the catalytic current with Fe NTA in a dose dependent manner (Fig. 6.3B). The membrane resistance values calculated by EIS for the BRACE formed with OmcB were still within the 2 range of highly insulating bilayer (~1 MΩcm ). Therefore it is unlikely that Fe NTA would have leaked through the bilayer of BRACE to reach PpcA. The more likely explanation is that Fe NTA is getting reduced by the OmcB protein immobilized in BRACE and exposed to the solution side of BRACE. Our hypothesis is that during the cathodic wave of CV, electrons travel from the gold electrode, to PpcA and from PpcA to OmcB and from OmcB to ferric NTA. 74 -heme In order to investigate our hypothesis further, we immobilized PpcA protein on SAM using EDC and NHS coupling agent. The BRACE formed with these interfaces did not display any redox peaks associated with the PpcA protein (Fig. 6.4A). Also BRACE formed with -heme OmcB and PpcA did not display any catalytic activity of OmcB with Fe NTA. Thus, the presence of immobilized and active PpcA is crucial to form a electron transport chain in BRACE. In order to investigate importance of OmcB in BRACE, we replaced OmcB proteoliposomes with OmcBc1 proteoliposomes. The BRACEs formed with PpcA and OmcBc1 proteoliposomes did not display the catalytic current increase with soluble electron acceptor Fe NTA (Fig. 6.4B). This is consistent with the observation that cysteine 1 mutation in OmcB sequence causes it to become redox inactive (please refer to chapter 4). And the redox inactive OmcB protein is incapable of transferring electrons from PpcA to the Fe NTA located on the solution side of the BRACE. Thus in order to transfer electrons form a electron transfer chain in BRACE, both PpcA and OmcB need to be present in the active form. The feasibility of BRACE to characterize ion channel activity was evaluated using gramicidin. Gramicidin is a pore forming antibiotic utilized to test the ion channel formation activity in bilayer lipid membranes. (230, 231) Gramicidin exists in a dimeric form with monomers in upper and lower leaflet, and once aligned, these monomers assemble to form a ion selective channel across the membrane. (232) Addition of gramicidin to the BRACE lead to channel formation in the lipid bilayer resulting a drop in the membrane resistance from 1.2 to 2 0.32 MΩcm (Fig.6.5). Addition of Fe NTA solution to this BRACE did not increase the 75 catalytic current of PpcA entrapped below the bilayer (data not shown). This observation is consistent with the ion selective nature of the gramicidin channels. 6.4 Implications and future work The biological cell envelopes have a very complex architecture in terms of chemical nature of the components involved as well as its multilayered structural arrangements. (233, 234) Several different types of lipids, carbohydrates and transmembrane proteins make them very complex and dynamic in nature. Therefore it is extremely challenging to characterize the mechanism of the metabolic processes taking place through them. Membrane proteins associated with the biological membranes play a crucial role in these metabolic processes. (235, 236) Membrane proteins constitute over one third of all proteins encoded by the bacteria, (237) yet very little information is available about the role of individual proteins. Because of the difficulties associated with the study of membrane proteins in the living organisms, it is preferable to study the proteins using in vitro platforms. Formation of engineered lipid bilayer membranes facilitates the study of these membrane proteins in a more controlled environment. (238) Hybrid bilayer membrane (HBM) is one of such strategies used to study the membrane proteins on the electrode. (239) In HBM, first a SAM is formed on a gold surface and upper leaflet of the lipid bilayer is added by using liposomes. Such HBM structure has been used to study electron transfer process using redox active small biomolecules ubiquinone and NADH. (240) Other approaches to study electron transfer properties of the membrane proteins are its directional immobilization of proteins on the SAM coated electrode (241) or tethering the protein to electrode keeping it in the lipid environment. (242) All of these well designed strategies often focus on studying only one set of specific proteins or biomolecules. With the design of BRACE, we have gone one step forward, where mimic of cell envelope makes study of ET reaction 76 occurring through different cell envelope compartments possible. In the inner membrane of biological cell envelope, the ET takes place only through some specific redox moieties. Formation of SAM on the gold electrode blocks the access of electrode to some species in solution, forming a mimic of inner membrane. Immobilization of PpcA on SAM, allows direct electron transfer between protein and electrode, and it thus forms a mimic of periplasmic space. Addition of DOPC liposomes leads to formation of bilayer, and forming an insulating layer between immobilized protein and electron acceptors in solution. The DOPC bilayer thus forms a mimic of outer membrane of the bacterial cell envelope. Addition of OmcB through proteoliposomes, adds the dimensionality of outer membrane cytochromes. The cytochrome protein is capable of embedding in the lipid bilayer. (243) It is possible that immobilized protein PpcA has similar interaction with the deposited liposomes, which might bring the OmcB in the proteoliposomes closer to PpcA in a manner to make ET possible. Because of the low concentration of OmcB in the proteoliposomes, we do not observe a change in CV of immobilized PpcA, but addition of soluble electron acceptor may augment the signal of OmcB. Similar increase of signal was reported for the cytochrome c oxidase immobilized on the electrode, where no redox current of cytochrome c oxidase was observed, but addition of its redox partner cytochrome c in solution led to increase in observable catalytic current. (244) Moreover, with the use of inactivated mutant of OmcB (OmcBc1), we have shown that the increase in the catalytic current is dependent on the activity of OmcB. Furthermore, replacement of PpcA with inactive polypeptide of PpcA also led to diminishing of ET, underlining importance of both the redox proteins in ET through BRACE. Formation of ion channels with gramicidin provides an interesting opportunity to add, small porins to the BRACE to mimic the cell envelope even closely. 77 Thus we have engineered an electron transport chain on artificially constructed mimic of cell envelope using BRACE. A schematic picture of the BRACE comparing it with the components of Geobacter cell envelope is drawn in Figure 6.6. Further structural characterization studies will help in optimization of BRACE formation. Also, more studies about optimization of proteoliposomes addition to maximize the loading of OmcB or outer membrane protein to BRACE will increase its sensitivity. The BRACE is a generic system that can be used with different membrane cytochromes, or even porins to test hypotheses about electrochemical membrane phenomena in gram negative microbes. The bottoms up approach used in the development of BRACE interface allows us to understand the ET at individual steps and build a more complex interface in sequential manner. Understanding dissimilatory metal reduction is a challenging task. The redox state of metallic electron acceptors depends on many factors including pH, presence of complexing agents, and the reduction potential values. The reduction potential values of U(IV)/U(VI) pair varies from 2+ 0.042 to 0.086 V (vs SHE) depending on the presence of Ca 2- and CO3 . (245) Similarly in the 2+ 3+ absence of precipitation, the midpoint potential values of Fe /Fe pair is about 0.77 V (vs. SHE); however at pH values > 3, the iron starts precipitating out of the solution and the redox 2+ potential values of Fe /Fe 3+ pairs under the environmental condition changes. Table 6.1 summarizes the redox potential values at pH 7 of species relevant to dissimilatory metal reduction in Geobacter. ∆G calculations are assumed on standard condition at pH 7, and for iron minerals, the Fe 2+ concentration is assumed to be 100 µM. (35) Calculations of the bioenergetics based on the redox potential values can help understand the ET mechanism in DRMs, while the BRACE approach can help test the feasibility of the ET between various redox carriers. 78 Successful fabrication of the functional, nanostructured biomimetic interfaces described above will enable hypotheses about electron transfer mechanisms in Geobacter to be tested, to achieve an improved fundamental understanding of Geobacter ET process. The interfaces could also potentially lead to better bioremediation strategies for heavy-metal contamination in the environment. 79 7.0 CONCLUSIONS The work presented in this dissertation mainly focused on developing a tool to measure the Geobacter ET mechanism in a more controlled, in-vitro environment. We utilized a bottom up approach to develop a nanoscale biomimetic interface necessary for such study using a self assembled monolayer of the lipids on electrodes, immobilized periplasmic protein PpcA, a lipid bilayer mimicking the outer membrane of the bacteria and an outer membrane protein OmcB. The first objective of this work was to establish feasibility of direct electron transfer (DET) between immobilized periplasmic cytochrome PpcA and the electrode. The Geobacter periplasmic cytochrome PpcA was expressed in E. coli to facilitate its large scale production necessary for the study. The heterologously expressed PpcA was isolated to obtain the pure protein. The purified protein was capable of DET with the electrode. The ratio of carboxyl to hydroxyl terminated lipids in the SAM used to immobilize the PpcA was found to be crucial for the activity of protein. In the second part of the study, we analyzed the catalytic activity of the immobilized PpcA using CV. The immobilized protein PpcA facilitated the electron transfer between the electrode and soluble electron acceptor species like iron and uranium salts/chelates. We fitted a literature-validated model to the CV data, and found that the reduction rates by PpcA are higher for uranium salts than iron salts. This observation may help explain why uranium gets rapidly precipitated in periplasmic space of Geobacter, where this PpcA cytochrome is present in abundance. We also found that the rotating disk voltammetry technique where the effect of mass transfer limitations is minimized enabling us to extract kinetic data can be used to calculate the ET rate constants between PpcA and soluble metal acceptors. 80 The third part of the study focused on heterologously expressing the Geobacter outer membrane cytochrome of OmcB in E. coli. We utilized a variety of molecular biology techniques, with the help of our collaborators, to transform E. coli cells with the plasmid carrying OmcB gene from Geobacter. Heterologous expression of outer membrane cytochromes in E. coli is challenging, because the cytochromes require extensive posttranslational modifications, including heme packaging. Moreover, the protein needs to be transported to the outer membrane and inserted in an active confirmation. We have been successful in expressing this multiheme cytochrome in E. coli, and based on the localization experiments such as differential detergent extraction and/or cell fractionation followed by ultracentrifugation, the cytochrome was found to be associated with the outer membrane of E. coli. To our knowledge, we are the only group to be successful in heterologous expression an outer membrane cytochrome from Geobacter in E. coli. The fourth part of this project focused on using spectro-electrochemical techniques in a optically transparent thin layer electrode (OTTLE) system to analyze electrochemical interactions between the PpcA and OmcB cytochromes. The results revealed that PpcA gave stable and reproducible DET in OTTLE and displayed an easily distinguishable UV spectroscopic signature. No evidence of DET by OmcB was observed in OTTLE. However, the addition of redox mediator (e.g., the humic acid analogue AQDS) allowed spectroelectrochemical characterization of OmcB in OTTLE. Experiments conducted with both PpcA and OmcB co-immobilized in the biomimetic interface showed that PpcA can act as a redox mediator for OmcB and is capable of reducing and oxidizing OmcB. This observation is significant because it supports the hypothesis that PpcA and OmcB are redox partners. A putative mechanism involving these proteins is that PpcA transmits electrons generated in respiration to OmcB, which then transmits the electrons to extracellular electron acceptors or 81 conductive pili. To our knowledge, this study provides the first evidence of direct electron transfer between these two cytochromes. Also, to the best of our knowledge, we are the first group to study protein-protein electron transfer in OTTLE. The final objective of this study was to develop a novel biomimetic redox active cell envelope (BRACE) to mimic Geobacter’s ET pathway with a minimum number of components. The BRACE consisted of an electrode to mimic the intracellular source of electrons, a SAM on the electrode to mimic the inner membrane, a layer of periplasmic cytochrome PpcA to mimic the periplasmic space, a bilayer lipid membrane to mimic the outer membrane and the outer membrane cytochrome OmcB added to the bilayer using proteoliposomes to represent outer membrane cytochromes. The BRACE thus formed was capable of transferring electrons from the electrode through the immobilized PpcA and then through the outer membrane cytochrome OmcB to the electron acceptors in solution. Fabrication of these nanoscale biomimetic interfaces has shown to have a great potential for study of electron transfer between redox proteins in vitro. Further characterization of these BRACE platforms can make them suitable for even diverse usage and as an important analytical tool. And the knowledge obtained from these systems can be helpful in devising better bioremediation technologies or designing a better microbial fuel system. 82 APPENDICES 83 APPENDIX A OMCB EXPRESSION AND PURIFICATION 84 A.1 Detergent solubilization of membrane proteins Membrane proteins like OmcB are difficult to study because they are generally insoluble in aqueous solution. Because of their high hydrophobicity they are difficult to work with. Moreover, membrane proteins are naturally embedded in or are strongly associated with a lipid bilayer, which is a complex and dynamic environment. Therefore, the biophysical studies of the protein such as circular dichroism, NMR, X-ray crystallography and ligand binding studies are difficult to perform.(157) Detergents are an important tool for studying the membrane proteins. Detergents are amphipathic in nature with a lipid tail group and a polar head group. Above a threshold concentration of detergents, called critical micelle concentration (CMC), the detergent self associate and form micelles. Above the CMS, the detergents are capable of solubilizing lipids and membrane proteins. (158) The head group of protein can impart cationic, anionic or zwitterionic properties to the detergent, and in general has stronger influence about its interaction with protein. While the tail group of detergent determine CMC. (159) We attempted solubilization of heterologously expressed OmcB with a variety of detergents at different concentration. The list of name of the detergents and concentrations is give in table A.1. Unfortunately, none of the conditions tried could result in the stable solution of the protein. Change of the host cell was tried as a mechanism to get better solubility of expressed protein. Instead of BL21 (DE3) E. coli cells, Origami2 TM TM and Origami B cells were used. Origami strains carry mutation in thioredoxin reductase and glutathione reductase gene, which result in better disulfide bond formation in cytoplasm resulting in higher active protein.(160) Origami B cells are derivative of Bl21 cells. The origami cells were transformed with pEC86 plasmid, and were selected on chloramphenicol plates. The selected cells were grown and made electrocompetent using previously published protocols.(161) The competent cells were 85 electroporated with OmcB plasmids, to transform the DNA in Origami cell lines. The transformed cells were selected on media containing amp and cmp. The colonies were picked and used for production of OmcB. However the OmcB expressed in these host cell line didn't have better solubility profile. A.2 Transmission electron microscopy (TEM) of E. coli cells Possibility of inclusion body formation due to overexpressed recombinant protein was tested by TEM. 1 mg/ml suspension of E. coli was fixed with gluteraldehyde in 50 mM phosphate buffer and post fixed wit Osmium tetroxide. The post fixed samples were dehydrated with a series of alcohol solutions, infiltrated and embedded with Araldite resin. Sections were taken with glass knife and ultrathin sections were taken with microtome. The ultrathin sections were mounted on the grid and images were taken using a transmission electron microscope (TEM, JEOL 100CX, Peabody, MA) operated at 50 K, 67 k, 100 K, and 200 K accelerating voltage. The E. coli cells expressing OmcB did not show any deposits in the periplasmic space ruling out formation of inclusion body type complexes (Fig. A.1). A.3 Addition of maltose binding protein affinity tag Maltose-binding protein (MBP) is one of the most popular fusion protein tag added to protein to aid in the affinity purification of protein with gentle elution conditions.(162) Another advantage associated with MBP fusion protein is that it aids in increasing solubility of the overexpressed protein. pMAL™ protein fusion and purification system uses the pMAL vectors designed to insert the target gene results in an MBP fusion protein. pMAL p5G vector was chosen because of its ability to deliver the fusion into the periplasmic space. pMAL p5G vector and OmcB genes were cut with NdeI and EcoRI restriction enzyme and the cut vector and insert 86 were ligated together. The ligation product was transformed with DH5 α cells and purified plasmid was used to co transform BL 21 cells. The SDS PAGE of the cells showed a band at expected molecular weight of 125 kD (~ 84 kD for OmcB+ 42 kD for MBP) in induced cells. The cells were harvested and the membranes were separated by the method described previously. The fusion protein was found to be membrane associated (Fig A.2). However the fusion protein was still very resistant to solubilization by detergent treatment. A.4 Gel filtration column chromatography under denaturing conditions Chaotropic salts like urea were utilized to solubilize OmcB under denaturing condition. The OmcB protein was purified by gel filtration column chromatography using a superdex TM 200 column. The flow rate was 1 ml/min. The elution was monitored by UV and the fractions were collected and subjected to SDS PAGE. The fractions with OmcB mol size were pooled together and renaturation was attempted by lowering the concentration of urea. (Fig.A.3) However the yield of this method was unsatisfactory and the renaturation of the protein was not easy to verify without the knowledge of structural characteristic information (like circular dichroism spectra) available. 87 APPENDIX B FIGURES AND TABLES 88 Figure 1.1: The pathways of energy metabolism conserved across different species of Geobacter. 89 Figure 1.2: The model for dissimilatory metal reduction by Geobacter. Energy is conserved by proton pumping across inner membrane. Electron transfer across the cell membrane takes place. 90 Figure 1.3: Transfer of electron across bacteria along the potential gradient. Redox potential of many outer membrane protein is yet unknown. 91 Figure 2.1: A. Chromatogram of PpcA capture step from the periplasmic fraction using cation exchange resin. Protein is eluted with a step gradient in salt concentration. B. Chromatogram of PpcA desalting by buffer exchange using gel filtration column. Protein is elutes first (monitored by UV signal) followed by salt (monitored by conductivity). 92 Figure 2.2: Purification of recombinant PpcA using cation exchange chromatography with a NaCl gradient. Inset, SDS-PAGE of PpcA elution peak containing the 9.6 kDa PpcA protein (arrow). Numbers at left are molecular weight markers in kDa. 93 Figure 2.3: Dithionite-reduced minus oxidized spectra of recombinant PpcA protein in Tris buffer (pH 8.0) showing three absorption bands at 420, 522, and 552 nm wavelengths that are typical of c-type cytochromes. 94 Figure 2.4: CV of PpcA in solution at SAM covered gold electrode. Cathodic current is plotted positive and anodic current is plotted negative. From the average of cathodic and anodic peak, the midpoint redox potential is calculated. 95 Figure 2.5: Cyclic voltammograms of PpcA in solution on a SAM coated gold electrode at increasing scan rates (arrow) of 10, 25, 50, 100, 150, and 200 mV/s (B). Inset shows the linear correlation between the cathodic peak current and the square root of scan rate for the PpcA at electrode. 96 Figure 2.6: A. Cyclic voltammograms of PpcA immobilized on a SAMfunctionalized gold electrode at increasing scan rates (arrow) of 25, 50, 75, 150, 300, 400, 500 and 700 mV/s B. Inset shows the linear correlation between the cathodic peak current and the scan rate for the PpcA biomimetic interface. 97 Figure 2.7: Trumpet plot obtained from the change in cathodic and anodic peak potential at various scan rates in CV experiments. The data is fitted to Laviron model to get the ET rate constants for the protein and electrode 98 Figure 3.1: Conceptual design of the biomimetic interface. The SAM covered gold electrode serves as a mimic of an inner membrane (IM), the immobilized protein PpcA serves as a mimic of periplasmic space (PS), and the DOPC bilayer is a mimic of an outer membrane (OM). 99 Figure 3.2: Cyclic voltammograms of PpcA interfaces in the presence (black) or absence (grey) of 200 µM Fe-NTA (A) or U acetate (B) collected at a scan rate of 500 mV/s. Insets show similar CV curves but using DOPC interfaces (PpcA interface covered with a DOPC synthetic lipid bilayer) with (black) or without (gray) 600 µM Fe-NTA (A) or 400 µM U acetate (B). 100 Figure 3.3: Calculation of second order rate constants ko’ for Fe-NTA reduction by cyclic voltammetry. (A) Linear plot between kinetic parameter (phi, ϕ) and the inverse of the scan rate (V/s) for Fe-NTA concentrations between 800 and 2500 µM (legend). The slope of the o o regression line (K ) was used to calculate the Kf constant using Eq. 1, described in the Materials o and Methods. (B) Linear correlation between the Kf constant and the Fe-NTA concentration, whose slope corresponds to the second order rate constant ko’. 101 Figure 3.4: Rate constant values (ko’) of PpcA immobilized on SAM-functionalized gold electrodes for different iron and uranium electron acceptors calculated from CV curves. Significant differences (p < 0.05 or < 0.005) in t-test pairwise comparisons with Fe NTA are indicated with one or two stars, respectively. 102 Figure 3.5: RDE voltammogram of PpcA immobilized on a SAM-functionalized gold electrode in the presence of 400 µM Fe-NTA at different rotation speeds (200, 400, 800, 1200, 1500, 1800 and 2200 rpm). The electrode potential was maintained at -500 mV against Ag/AgCl. 103 Figure 3.6: (A) Linear correspondence between cathodic currents (reverse of steady state current values or iss) of PpcA immobilized on a SAM-functionalized rotating disc gold -1/2 electrode and the rotations speed (ω ) for different concentrations of Fe-NTA (in µM). (B) Linear correspondence between limiting current (ilim) calculated from (A) and Fe-NTA concentration used to calculate the second order rate constant value ko’ 104 Table 3.1: ko’ values for different metal acceptors obtained by cyclic voltammetry and rotating disk electrode method. Metal species ko’from CV 6 ko’ from RDE (x10 M s ) -1 -1 (x106 M s ) Ferric NTA 0.056 ± 0.020 0.052 ± 0.010 Ferric citrate 0.025 ± 0.010 0.012 ± 0.001 Ferric nitrate 0.103 ± 0.100 0.061 ± 0.010 Uranyl acetate 0.786 ± 0.180 NDa Uranyl nitrate 0.990 ± 0.240 ND a Not determined 105 -1 -1 Figure 4.1: OmpA-OmcB Amino acid sequence. Peptide sequences identified by MS are highlighted in blue. MS identified 23 unique peptides for OmcB in 151 total spectra. 380 out of 742 amino acids identified resulting in 51% coverage. 106 Figure 4.2: SDS PAGE profile of OmcB expression and cell fractionation. Lane 1and 2 are fermentation media samples normalized for cell density at 3 hrs and 18 hrs after inoculation. Lane 3 and 4 are crude and clarified cell lysate. Lane 5 is supernatant of ultracentrifugation step, lane 6 is membrane pellet. Lane 7 is supernatant of ultracentrifugation after sarkosyl detergent extraction and lane 9 is OM fraction lane 8 is a molecular weight marker with molecular weights shown on right in kD. 107 Figure 4.3: Ultracentrifugation with sucrose density gradient separates OmcB membrane fractions based on buoyant density. IM fractions are retained in 37% sucrose band, while OM fractions are located at intersection of 60 and 70% sucrose. 108 Figure 4.4: Whole cell spectroscopy of E. coli BL21 cells expressing OmcB. Blue line is spectra is air oxidized cells at ~ 1 mg/ml. Red Spectra showing Soret peak at 420 nm is spectra of sodium dithionite reduced cells, in some spectra the α peak at 552 nm is visible. Black spectra is spectra of reoxidized cells (observed by shift in Soret peak) with A. FeNTA, B. ferric citrate, C. riboflavin and D. ferric oxide particles. 109 Figure 4.5: OM fragments containing OmcB reduced different metal samples. Blue spectra is air oxidized OmcB samples, red spectra is sodium dithionite reduced OmcB, and black spectra is OmcB reduced with A. FeNTA, B. Ferric citrate, C. UA, and D. Ferric oxide particles. 110 Figure 4.6: Cysteine mutation at 1 cysteine to alanine affected redox property of the OmcB. Spectra of oxidized (solid line) and sodium dithionite reduced (dashed line) OmcBc1a mutant showed peaks not related to cytochrome c protein. 111 Table 4.1: List of different plasmids used for OmcB expression in E. coli. Primer Sequence Plasmid 5’OmpAACCATATGAAAAAGACAGCTATCGCGATTGCAG pET21c C- OmcB TGGCACTGGCTGGTTTCGCTACCGTAGCGCAGG His6 5’ NdeI CCTGCGGCTCCGAAAACAAGGA-3’ 5’- OmcB XhoI 3’ His fusion GTAAGTCTCGAGCGGACGGGTCGTGCCGAG GTA-3’ 5’- OmcB 5’ pET22b CAATACCATGGGCTCCGAAAACAAGGAGGGGAC- NcoI His6 3’ 5’- OmcB 3’ ATATCTCGAGCGGACGGGTCGTGCCGAGGTAGXhoI 3’ OmcB Cys mut 5’ OmcB Cys mut 3’ 5’GTAGCGCAGGCCGCCGGCTCCGAAAAC-3’ 5’GTTTTCGGAGCCGGCGGCCTGCGCTAC-3’ 112 pET21c CHis6 mut Figure 5.1: A. Schematic diagram of an OTTLE cell. The spacer (SP) separating the transparent window and the working electrode (WE) forms a thin layer (TE), which determines the volume of the cell. The counter or auxiliary electrode (AE) is connected to the TL via a small capillary containing supporting electrolyte (EC). Reference electrode (RE) is loaded through the loading port (LP). B. Schematic picture of the working electrode. 113 Figure 5.2: Square wave voltammogram of PpcA adsorbed on SAM coated transparent gold electrode. The protein response was stable for 6 hr (Solid line 1 hr and dashed line 6 hr), and the peak current decreased around 20% after 24 hr (dotted line). 114 Figure 5.3: A. Cyclic voltammograms of PpcA in solution on a SAM coated gold electrode at OTTLE with increasing scan rates of 5, 10, 25, 50, and 100 mV/s B. Inset shows the linear correlation between the cathodic peak current and the square root of scan rate for the PpcA at the OTTLE. 115 Figure 5.4: UV-vis spectroscopic response of PpcA at the OTTLE (A) Difference absorption spectra oxidized (blue) and reduced (red) PpcA (B) Reduced minus oxidized spectra of PpcA at different electrode potentials (vs. Ag/AgCl) showing wavelengths of interest that can be monitored as a function of redox state. 116 Figure 5.5: A. Chronoamperometry of PpcA at OTTLE. Kinetics of the decrease in absorbance at 552 nm follow the reduction current decay. B. Reproducibility of PpcA reduction at the SAM coated OTTLE show reversible and stable response of cytochrome. 117 Figure 5.6: Subtraction spectra of OmcB with mediator (AQDS 50 µM) at OTTLE during the CV run at 1 mV/s. Changes in absorbance at wavelengths characteristic of cytochrome c proteins are observed. 118 Figure 5.7: Spectroelectrochemical characterization of OmcB heme potentials using redox mediators. Change in the absorbance at 410 nm (square marker) is followed as a function of the potential (red steps). Clustering of the absorbance data at particular electrode potentials may be contributed by resolution of individual heme groups. 119 Figure 5.8: AQDS mediated reduction of OmcB membrane vesicles at OTTLE monitored by spectroscopic technique. Potential of electrode was shifted in increments of 50 mV and held at that potential for 180 sec. 120 Figure 5.9: Cyclic voltammogram of PpcA with and without OmcB membrane vesicles at 50 mV/s. A small increase in the reduction current of the protein may be due to transfer of electrons from PpcA to OmcB. 121 Figure 5.10: Spectroscopic response of OmcB reduction mediated by PpcA. A. Spectra of reduction and oxidation of PpcA with and without OmcB membrane vesicles. B. Redox spectra of OmcB obtained from subtracting spectra of PpcA from the spectra of both proteins together in multipotential step voltammetry. 122 Figure 6.1: A. TEM images of DOPC liposomes prepared by sonication, taken with JEOL transmission electron microscope with accelerating voltage of 270 K. B. QCM study showing formation of lipid bilayer on the SAM. Attachment of liposomes to the substrate, initially decreases the vibrational frequency. Rupture of liposomes to release trapped water increases the frequency. 123 Figure 6.2: A. The phase angle and log z data plotted against log frequency for the bilayer made from proteoliposomes. The data was fitted to the equivalent circuit model shown in figure B. B. Randles equivalent circuit model used to fit EIS data to calculate the values of electrochemical properties. RS is the solution resistance, Cm is the capacitance of the membrane, Rm is the resistance of membrane and CPE represents the constant phase element. 124 Figure 6.3 A. CV of BRACE formed with PpcA and DOPC bilayer. The redox peak of PpcA is noticed (solid line), and addition of soluble electron acceptor Fe NTA (1mM) into solution does not increase the reduction current (dashed line), indicating formation of a blocking layer. B. CV of BRACE formed with PpcA and OmcB proteoliposomes. The redox peak of PpcA is evident (solid line), addition of Fe NTA increased the catalytic current in a dose dependent fashion: 1 mM (dotted line), 2 mM (dashed line) and 3 mM (dashed and dotted line). 125 -heme Figure 6.4 A. CV of BRACE formed with PpcA and OmcB proteoliposomes. The redox peak of PpcA is absent (solid line), and addition of soluble electron acceptor Fe NTA (1mM) into solution does not increase the reduction current (dashed line). B. CV of BRACE formed with PpcA and OmcBc1 proteoliposomes. The redox peak of PpcA is evident (solid line), but addition of Fe NTA into solution does not increase the reduction current. 126 Figure 6.5: Measurement of gramicidin ion channel activity at BRACE using EIS. Impedance data for BRACE is shown before (hollow square and triangles) and 1 hr after addition 1 µM gramicidin . Drop in the resistance is evident due to the passage of sodium ions through BRACE. 127 Figure 6.6: Schematic representation of Geobacter cell envelope (A) and Biomimetic redox active cell envelope (B). A comparison can be made between inner membrane of the bacteria and SAM, periplasmic space cytochromes and immobilized PpcA, outer membrane and DOPC bilayer, and outer membrane cytochromes in Geobacter and OmcB deposited as proteoliposomes. (Figure not drawn to the scale) 128 Table 6.1: Reduction potentials and free energies of redox compounds Eenv* (V) Reduction pair ∆G (KJ/mol) Terminal electron acceptor Fe3+/Fe2+ (pH 2) +0.77 -74.2 Fe (III)-citrate/Fe (II) citrate +0.385 -37.1 Fe(III)-NTA/Fe(II)-NTA +0.372 -35.9 +0.1 to -0.1 -9.6 to 9.6 α-FeOOHsolid/Fe2+ -0.274 26.4 α- Fe2O3solid/Fe2+ -0.287 55.4 Fe3O4solid/Fe2+ -0.314 60.6 Ferrihydritesolid/Fe2+ Electron donors CO2/acetate -0.29 223.8 NAD+/NADH -0.32 61.7 Electron carriers Menaquinones (ox/red) -0.067 to -0.11 12.9 to 21.2 Ubiquinone (ox/red) +0.11 -21.2 PpcA -0.169 16.3 OmcS -0.212 18.3 OmcB ~ -0.18 * Eenv : Environmentally relevant redox potentials at pH 7, except where noted 129 Table A .1: List of some of the detergents tried to solubilize OmcB Detergent Detergent class Solubility n-Octyl-D- Non ionic No glucopyranoside (OG) detergent 0.5%, 1%, 1.5 %, 2% pH 3.5 and pH 8 Dodecyl b D maltoside (DDM) Non ionic No detergent 1%, 2% No Polyoxyethylene 1%, 2% Cell lyticTM detergent No mix CHAPS 1%, 2%, 4% Brij 35 1% Zwitterionic No detergent Non ionic No detergent MEGA-8 Non ionic No detergent MEGA 9 2% Non ionic detergent 130 No Table A.4.1 (cont'd) MEGA 10 2% Non ionic No detergent Sulfobetaine SB 10 2% Zwitterionic No detergent Sulfobetaine SB12 2% Zwitterionic No detergent Zwittergent 0.5%, 1%, 2%, 5% Zwitterionic No detergent Sodium deoxycholate Anionic detergent No Cetylpyridinium Cationic detergent Slight Cationic detergent Moderate chloride Cetyltrimethylammoni um bromide (CTAB) 2% DDM2% + 6 M Urea Moderate OG 2% +6 M Urea Moderate OG 2% + 200 mM No Urea 131 Figure A.1:A, B: TEM images of E. coli BL 21 cells expressing OmcB protein. 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