new approaches for fresh perspectives on quinol/quinone oxidoreductases

Lead Research Organisation: University of East Anglia
Department Name: Chemistry

Abstract

All living cells are surrounded by a thin membrane that shields and separates the inside of these cells from their surroundings. In more advanced organisms, organelles are located inside the cell with specific functions. Also these organelles are separated (compartmentalised) from the rest of the cell by membranes. These thin membranes contain many proteins that actively transport compounds, like nutrients and salt, specifically across the membrane. Consequently, the concentration of many compounds is different on the inside of the membrane compared to the outside. These gradients play a crucial role in biology and many reactions in the cell are dependent on them, like photosynthesis and metabolism. Some of these proteins actively 'pump' protons across the membrane using energy that is released from electrons that are formed when sugars and fats are 'burned' by the cell. These electrons do not flow freely in the cell, but are attached to small molecules which 'float' in the membranes of the cell. These molecules are called quinones or co-enzyme Q. This proposal aims to develop a new tool with which we can study the proteins that are located in the membrane and react with quinones. Why do we want to learn more about these proteins? These proteins are involved in many important reactions. For instance, in bacteria they are responsible for all reactions involving nitrogen and carbon dioxide and therefore control how these elements are recycled in our atmosphere. In humans, similar proteins are involved in the burning of sugars and fat and the production of energy; Any problems with these proteins and we become ill. Finally, quinones themselves are 'anti-oxidants' and known to take away so-called 'radicals' which are thought to play an important role in diseases and aging. When we study the structure and function of proteins and quinones in the lab, they are normally taken out of the membrane and thus the environment of these proteins and quinones is changed a great deal. This is done because membranes do not dissolve in water and most of our experiments are performed in water; we thus need to take the membrane away. However, in this proposal we aim to develop a new tool that allows the study of membrane proteins and the quinone in their natural environment, the membrane. For this to be achieved, we will first place a 'membrane protein' that normally receives or gives electrons to the quinones on a solid surface. This solid is conducting (like metal wires) and we will carefully control the properties of the surface so that it will be possible to give or take electrons to or from the protein. We will then place a membrane on top of the proteins and this membrane will contain quinones. If everything works as we think it will, the protein will give or take electrons to or from the quinones. As the transfer of electrons is nothing more than electrical current, we can measure very accurately how fast these electrons are passed from the surface to the proteins and into the quinones (or the other way around). Once this system is complete, we can use these surfaces to 'interrogate' these membrane proteins in almost the same membrane environment they encounter in the cell. By studying these proteins we will thus learn more about how they function inside their natural membrane.

Technical Summary

Quinone oxidoreductases (QORs) play central roles in respiration and photosynthesis with additional roles in anti-oxidant production and biosynthesis. However, when compared to our understanding of globular enzymes, relatively little is known about the kinetics of enzyme-catalysed QH2 ? Q transformations. In large part this is a consequence of constraints on diffusion and solubility that result from membrane environments that have proved difficult to mimic and manipulate reproducibly until recently. Here, we will combine two 'tools' - solid-supported membrane technology and protein-film voltammetry - to develop new methods for quantitative analysis of QOR catalysed transformation of lipophylic substrates. Studies with members of a widespread family of respiratory QH2 dehydrogenase will provide proof of principle for methods that will be ultimately be applicable to many QORs. Three strategies will be compared for their ability to support electrochemical characterisation of QOR activity in a membrane environment supported by a graphite or gold electrode, i) proteoliposome adsorption, ii) vesicle adsorption on adsorbed protein films and iii) vesicle fusion, i.e., planar membrane formation, on adsorbed protein films. Voltammetry will define the stability and electroactive coverage of the films, the reduction potentials and interfacial electron transfer rates for the adsorbed proteins and their QOR activities. Where films are formed on gold electrodes complementary information will be provided from other methods. Surface plasmon resonance, atomic force microscopy and quartz crystal microbalance methods will define the total amount of adsorbed protein for comparison to the amount of electroactive protein. Surface-enhanced resonance Raman spectroscopy and/or surface-enhanced Fourier transform infrared spectroscopy will inform on structural differences between assemblies.

Publications


10 25 50


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Cheetham MR (2011) Concentrating membrane proteins using asymmetric traps and AC electric fields. in Journal of the American Chemical Society

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Doyle RM (2013) Contrasting catalytic profiles of multiheme nitrite reductases containing CxxCK heme-binding motifs. in Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry





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Maalcke WJ (2014) Structural basis of biological NO generation by octaheme oxidoreductases. in The Journal of biological chemistry


 
Description All living cells are surrounded by a thin membrane that shields and separates the inside of these cells from their surroundings. In more advanced organisms, organelles are located inside the cell with specific functions. Also these organelles are separated (compartmentalised) from the rest of the cell by membranes. These thin membranes contain many proteins that actively transport compounds, like nutrients and salt, specifically across the membrane. Consequently, the concentration of many compounds is different on the inside of the membrane compared to the outside. These gradients play a crucial role in biology and many reactions in the cell are dependent on them, like photosynthesis and metabolism. Some of these proteins actively 'pump' protons across the membrane using energy that is released from electrons that are formed when sugars and fats are 'burned' by the cell. These electrons do not flow freely in the cell, but are attached to small molecules which 'float' in the membranes of the cell. These molecules are called quinones or co-enzyme Q.

This proposal developed a new tool with which we can study the proteins that are located in the membrane and react with quinones. Why do we want to learn more about these proteins? These proteins are involved in many important reactions. For instance, in bacteria they are responsible for all reactions involving nitrogen and carbon dioxide and therefore control how these elements are recycled in our atmosphere. In humans, similar proteins are involved in the burning of sugars and fat and the production of energy; Any problems with these proteins and we become ill. Finally, quinones themselves are 'anti-oxidants' and known to take away so-called 'radicals' which are thought to play an important role in diseases and aging.

When we study the structure and function of proteins and quinones in the lab, they are normally taken out of the membrane and thus the environment of these proteins and quinones is changed a great deal. This is done because membranes do not dissolve in water and most of our experiments are performed in water; we thus need to take the membrane away. Here we developed a new tool that allows the study of membrane proteins and the quinone in their natural environment, the membrane. For this to be achieved, we placed a 'membrane protein' that normally receives or gives electrons to the quinones on an electrode surface. This solid is conducting (like metal wires) and was tailored to allow electrons to be exchanged directly between the electrode and the protein. A membrane containing quinones was placed on top of the proteins in such a way that the protein exchanged electrons with the quinone. These transfers of electrons give rise to an electrical current that we can meaure to quantify how fast these electrons are passed from the surface to the proteins and into the quinones (or the other way around).

We have used this method to 'interrogate' membrane proteins in almost the same membrane environment they encounter in the cell. For one of these proteins, purified from a microbe, we have discovered that electrons only flow in the direction required for the cellular activity when the membrane protein is docked to a soluble protein. This has important consequences for the design of microbial 'factories'. These are microbes that are engineered to harness electron transfer reactions for the synthesis of renewable chemicals, carbon-capture and the harnessing of bioenergy in microbial fuel cells in developments that are set to address our overdependence on fossil fuels and polluted atmosphere.
Exploitation Route The finding that the quinol dehydrogenase of Shewanella is selective for reduction of menaquinone has implications for those developing microbial fuel cells and strategies for microbial electrosynthesis. Electron transfer through the quinol dehydrogenase is key to electron exchange between cytoplasmic processes and external electrodes and both industrial processes require greater knowledge of the biochemistry/biophysics underpinning this event and as provided as an outcome of this grant.
Sectors Energy,Manufacturing, including Industrial Biotechology
 
Description - training and development of staff employed on grant. - science communication events delivered by staff on grant.
First Year Of Impact 2010
Sector Energy,Environment
Impact Types Cultural,Economic
 
Description EPSRC responsive mode
Amount £313,231 (GBP)
Funding ID EP/M001989/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 01/2015 
End 12/2017
 
Description Royal Society Leverhulme Trust Senior Research Fellowship
Amount £54,000 (GBP)
Organisation The Royal Society 
Department Royal Society Leverhulme Trust Senior Research Fellowship
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2015 
End 09/2016
 
Description PNNL DoE support 
Organisation U.S. Department of Energy
Department Pacific Northwest National Laboratory
Country United States of America 
Sector Public 
PI Contribution Protein purification, spectroscopic and electrochemical analysis of purified proteins, biochemical assay of purified proteins
Collaborator Contribution provision of bacterial strains overexpressing target proteins
Impact The X-ray crystal structure of Shewanella oneidensis OmcA reveals new insight at the microbe-mineral interface. Edwards MJ, Baiden NA, Johs A, Tomanicek SJ, Liang L, Shi L, Fredrickson JK, Zachara JN, Gates AJ, Butt JN, Richardson DJ, Clarke TA. FEBS Letters 2014 in press Rapid electron exchange between surface-exposed bacterial cytochromes and Fe(III) minerals White GF, Shi Z, Shi L, Wang L, Dohnalkova AC, Marshall MJ, Fredrickson JK, Zachara JN, Butt JN, Richardson DJ, Clarke TA PNAS 2013 110 6346 - 635 Molecular structure and free energy landscape for electron transport in the decahaem cytochrome MtrF Breuer M, Zarzycki P, Shi L, Clarke TA, Edwards MJ, Butt JN, Richardson DJ, Fredrickson JK, Zachara JM, Blumberger J and Rosso KM Biochem. Soc. Trans. 2012 40 1198-1203 The roles of CymA in support of the respiratory flexibility of Shewanella oneidensis MR-1 Marritt SJ, McMillan DGG, Shi L, Fredrickson JK, Zachara JM, Richardson DJ, Jeuken LJC and Butt JN Biochem. Soc. Trans. 2012 40 1217-1221 Development of a proteoliposome model to probe transmembrane electron-transfer reactions White GF, Shi Z, Shi L, Dohnalkova AC, Fredrickson JK, Zachara JM, Butt JN, Richardson DJ and Clarke TA Biochem. Soc. Trans. 2012 40 1257-1260 The crystal structure of the extracellular 11-heme cytochrome UndA reveals a conserved 10-heme motif and defined binding site for soluble iron chelates Marcus J, Edwards, Andrea Hall, Liang Shi, James K. Fredrickson, John M. Zachara, Julea N. Butt, David J. Richardson, and Thomas A. Clarke Structure 2012 20 1275-1284 Exploring the biochemistry at the extracellular redox frontier of bacterial mineral Fe(III) respiration Richardson, DJ, Edwards, MJ ; White, GF Baiden, N; Hartshorne, RS; Fredrickson, J; Shi, L; Zachara, J; Gates, AJ; Butt, JN; Clarke, TA Biochem. Soc. Trans. 2012 40 493-500 The 'porin-cytochrome' model for microbe-to-mineral electron transfer Richardson DJ, Butt JN, Fredrickson JK, Zachara JM, Shi L, Edwards MJ, White G, Baiden N, Gates AJ, Marritt SJ, Clarke TA Mol. Micro. 2012 85 201-212 A Functional Description of CymA that is the Hub for Anaerobic Respiratory Flexibility in Shewanella. Marritt SJ, Lowe TG, Bye J, McMillan DGG, Shi L, Fredrickson J, Zacchara J, Richardson DJ, Cheesman MR, Jeuken LJC, Butt JN Biochem. J. 2012 444 465-474 Identification and Characterization of MtoA: a Decaheme c-Type Cytochrome of the Neutrophilic Fe(II)-oxidizing Bacterium Sideroxydans lithotrophicus ES-1 Liu J, Wang Z, Belchik SM, Edwards MJ, Liu C, Kennedy DW, Merkley ED, Lipton MS, Butt JN, Richardson DJ, Zachara JM, Fredrickson JK, Rosso KM, Shi L Frontiers in Microbiological Chemistry 2012 3 1-11 Structure of a bacterial cell surface decaheme electron conduit. Clarke TA, Edwards MF, Gates AJ, Hall A, White GF, Bradley J, Reardon CL, Shi L, Beliaev AS, Marshall MJ, Wang X, Watmough NJ, Fredrickson JK, Zachara JM, Butt JN, Richardson DJ Proc. Nat. Acad. Sci. USA 2011 108 9384-9389 Characterization of an electron conduit between bacteria and the extracellular environment. Hartshorne RS, Reardon CL, Ross D, Nuester J, Clarke TA, Gates AJ, Mills PC, Fredrickson JK, Zachara JM, Shi L, Beliaev AS, Marshall MJ, Tien M, Brantley S, Butt JN, Richardson DJ Proceedings of the National Academy of Sciences 2009 106 22169-74
 
Description USC Tubulation of Proteoliposomes 
Organisation University of Southern California
Country United States of America 
Sector Academic/University 
PI Contribution Intellectual contribution, skills in (proteo-)liposome preparation and protein handling, purified proteins, training of research students and post doctoral researchers.
Collaborator Contribution Intellectual contribution, access to and experience with microscopies (white light, fluoresence, atomic force, transmission electron and cryo-electron) and measurement of the electrical conductivity of bacterial nanowires spanning interdigitated electrodes, training of research student and project supervisor in aforementioned methods.
Impact studies ongoing, too early for outputs
Start Year 2016
 
Description BBC Radio Norfolk interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact Broadcast Interview to highlight advances in understanding structures that conduct electrons through proteins. Impact was raised profile of our research and University.
Year(s) Of Engagement Activity 2014
 
Description UEA Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact interactive demonstrations run throughout the day prompted ongoing discussions with visitors about our research.

many people noted the research was fascinating and they were not aware of it previously
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014