new approaches for fresh perspectives on quinol/quinone oxidoreductases

Lead Research Organisation: University of Leeds
Department Name: Institute of Membrane & Systems Biology

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
publication icon
Bawazer L (2018) Enzymatically-controlled biomimetic synthesis of titania/protein hybrid thin films in Journal of Materials Chemistry B

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

publication icon
McMillan DG (2012) Menaquinone-7 is specific cofactor in tetraheme quinol dehydrogenase CymA. in The Journal of biological chemistry

 
Description In respiration and photosynthesis, a key role is played by enzymes that reduce and oxidise membrane-bound quinones. Due to the hydrophobic nature of these membrane enzymes and their substrates, the enzyme mechanisms has been hard to establish. We have used a relative simple quinone-converting enzyme from a Shewanella species, CymA, to study this quinone converstion with an electrochemical technique in which CymA is adsorbed on an electrode in such a way that electrons are exchanged between CymA and the electrode. This system enables the characterisation of lipohilic quinone turn-over, where the catalytic rate is measured directly as current (= electrons consumed or produced per second).

A key finding has been that CymA uses its substrate (menaquinone-7) in a co-factor like function. One menaquinone-7 remains bound to the enzyme where it functions in turning-over other quinones. While CymA is specific for menaquinone in its co-factor role and loses activity if menaquinone-7 is removed, it exhibits less specificity for its substrate as it also turns over ubiquinone and the water-soluble menadione. These results proof to be an important warning to crystallographers, who generally interpret a bound quinone or quinone-homologue in their protein structures as a substrate interacting with an enzyme. Overall, this key finding implies that enzyme catalysis in lipid membranes of lipohilic substrates has different properties compared to globular enzymes.

A second key finding is that the purified CymA is very strongly biased to a catalytic direction opposite to its in vivo function. The question arrose whether this is due to the purified enzyme being solubilised in a detergent solution (i.e., an artefact of the purification procedure) or whether the bias is controlled by binding to one of the many partner proteins of CymA to which, in vivo, CymA passes on electrons during respiration. To study this, CymA has been incorporated in a planar lipid membrane deposited on flat surface. CymA is present in two orientations: One with its large extra-membranous pointing towards to aqueous solution and one where this domain is wedged between the lipid membrane and the electrode surface. By applying a lateral electric field it was shown that only one of the two orientations is free to diffuse within the membrane. It is likely that when the extra-membranous domain is wedged between the surface and the planar lipid, CymA is unable to move.

The same planar membrane system was used to further characterise the catalytic properties of CymA. It was shown that when the redox partner FccA, a fumarate reductase, was added to the system, electron transfer was possible from Menaquinone-7 to CymA to FccA and finally fumarate. These data suggest that perhaps the catalytic bias of CymA is controlled by its interaction with redox partners.
Exploitation Route This project has contributed to our understanding of the respiration in bacteria, especially of Shewanella species. Shewanella is widely used as a model system to develop microbial fuel cells, which can generate energy out of a variety of fuel sources, including waste water.

A renewable energy cycle (including technologies that can reduce the energy cost of, for instance, waste water treatment) is recognized as a top national strategic priority in the UK (UK White Paper on Energy). At the time of finishing this project in 2013, several incidents demonstrated the fragility of the global energy supply: the sharp rise in oil prices following the outbreak of conflicts and civil wars in the Middle-East and the ecological and humanitarian threat of a nuclear meltdown in Fukushima, Japan. The search for alternative energy sources is therefore of major importance to our society. A solution to this problem has to be sought by combining a multitude of 'alternative' energy sources; this research will contribute to this progress. Shewanella species have formed an important model system for microbial fuel cells. Their respiration is typified by flexibility in fuel source and terminal electron acceptor. Important is their ability the exchange electrons with an electrode surface (the anode in the microbial fuel cell). CymA plays a central role in the respiration of Shewanella and fundamental knowledge of CymA is helping the MFC community in their research.
Sectors Energy,Environment

 
Description Advancing Microbial Electrochemistry: Biophysical Characterisation of the electron-transfer interactome in S. oneidensis MR-1
Amount £334,000 (GBP)
Funding ID BB/L020130/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2014 
End 10/2017
 
Description BBSRC Partnership Award
Amount £20,859 (GBP)
Funding ID BB/R020140/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2022
 
Description ERC Starting Fellowship
Amount € 1,650,829 (EUR)
Funding ID 280518 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 01/2012 
End 12/2016
 
Description University of East Anglia 
Organisation University of East Anglia
Country United Kingdom 
Sector Academic/University 
PI Contribution This award was a collaboration between us and the University of East Anglia. All details for the collaboration and research are described in detail in the research proposal.
Collaborator Contribution This award was a collaboration between us and the University of East Anglia. All details for the collaboration and research are described in detail in the research proposal.
Impact This partnership/collaborations has been funded by multiple BBSRC proposals. All outputs and outcomes are described in detail under the respective BBSRC awards.
Start Year 2009
 
Description University of Otago 
Organisation University of Otago
Department Department of Microbiology & Immunology
Country New Zealand 
Sector Academic/University 
PI Contribution We provided a research tool/methodology. The partner will visit the University of Leeds to use this research method.
Collaborator Contribution The partner will contribute a purified enzyme that will be studied as part of this collaboration.
Impact At the time of writing, experiments are still to be performed, so no outputs or outcomes have resulted yet. An BBSRC partnership award was applied for and funded (this information is provided under 'grants').
Start Year 2018