In 2011, the Jeuken group was awarded an ERC grant from the EU (FP7) for its project: Membrane-modified Electrodes for Membrane Enzymes (MEME). MEME, which ran from January 2012 - December 2016 and funded several PhD students, Post Doctoral fellows and a technician. A description of the main projects can be found below.
Electrochemical investigations of biological processes have provided a wealth of information on the structure-function relationship of redox enzymes, while the underlying technology has formed the basis for biosensors. Biosensors such as the extremely successful glucose biosensor have revolutionised the treatment of diabetes patients around the world and significantly improved their conditions. However, many of today's applications do not impose molecular control on the electrode-protein interaction, which limits their full potential in biosensing and impairs future applications in biological photo harvesting, biofuel cells and energy storage. While electrochemistry of globular redox-enzymes is limited by poor control of the surface-protein interface, the absence of control with membrane proteins has so far made it impossible to study them electrochemically. This in spite of the huge importance of membrane enzymes in biology and the great promise they hold in biosensing and energy generation. To solve these problems, the Jeuken Group has combined the state-of-the-art in surface physics, colloid and organic chemistry, membrane biology and electrochemistry to develop membrane-modified electrodes with full control of protein-electrode interactions. In this ERC Starting Grant proposal we will aim to consolidate this research by applying this methodology to hydrogenases and light-harvesting reaction centres, both of which have promising applications in biofuel cells. Second, we will show how the combination of our membrane-modified electrodes with fluorescence spectroscopy provides an exciting application in single enzyme research, a challenge that has been met for only a handful of membrane proteins. Single-enzyme kinetics of a proton-pumping haem-copper oxidase will provide new insights into the molecular mechanism of proton-pumping.
The long term vision of this work is to create electrodes which communicate with living cells. Such an interface would not only allow us to study bioenergetics in living cells, it would also pave the way to harness bioenergy in biofuel cell applications. In the final parts of this proposal, a first start in this ambitious vision is made by connecting our electrodes to living bacteria via their cytoplasmic membrane.
Hydrogenases have been of great interest over the last 10 years because of their potential application in biofuel cells as well as their intriguing enzymology. A sub-class of this family, the membrane-bound [NiFe]-hydrogenases, which oxidise hydrogen and donate electrons to the quinone pool, have been of specific interest as they are stable in oxygen atmosphere, an essential requirement for biofuel applications. However, being a membrane enzyme has made them difficult to study and the large majority of studies so far have been performed with only the two hydrophilic subunits (Hox GK, see Figure). However, the extraction of the hydrophilic subunits leads to a significant loss in activity and results in the loss of the quinone reducing activity as the quinone converting domain, HoxZ, is not present. In this project, we studied the complete heterotrimeric enzyme, both to further our insight into the catalytic mechanism of the enzyme and to evaluate applications in biofuel cell technology. This project was performed in collaboration with Prof. Oliver Lenz and Dr. Stefan Frielingsdorf from the Technical Univerity of Berlin. This German group has an extensive track-record in the study of hydrogenases from R. eutropha and other organisms.
The main outcomes of this project were:
1) Membrane-Bound [NiFe]-Hydrogenase (MBH) of Ralstonia eutropha was observed to display an enhanced tolerance towards oxygen when retained in a native-like lipid membrane. Previous electrochemical studies with this biocatalyst have always been performed with water-soluble subcomplexes, but our new insights indicate new ways in which this biocatalyst can be used in hydrogen-oxygen fuel cells.
2) The fact that we observed that MBH is less sensitive to oxygen in our systems (compared to previously published systems), together with the fact that our membrane-modified electrodes do not require purified enzymes but function with cellular extracts, might make these biocatalysts more suitable for hydrogen-oxygen fuels cells (manuscript in press in Adv. Funct. Mat.).
3) We obtained data that suggest that the oxygen tolerance of MBH is due to a electronic 'switch' mechanism, where the electron flow to and and from the active site of MBH is disconnected when oxygen is 'sensed' while no hydrogen is present.
Valentin Radu has now finished his studied and passed his viva in December 2016. He currently works at the University of Keele. This work was published in the following publications:
Heath, G.R., Li, M., Rong, H., Radu, V., Frielingsdorf, S., Lenz, O., Butt, J.N., Jeuken, L.J.C. (2017) Multilayered lipid membrane stacks for biocatalysis using membrane enzymes. Adv. Funct. Mat., 27, Art. No. 1606265. DOI: 10.1002/adfm.201606265
Radu, R., Frielingsdorf, S., Lenz, O., Jeuken, L.J.C. (2016) Reactivation from the Ni-B state in [NiFe] hydrogenase of Ralstonia eutropha is controlled by reduction of the superoxidised proximal cluster, Chem. Comm., 52, 2632 - 2635. DOI: 10.1039/C5CC10382G
Radu, V., Frielingsdorf, S., Evans, S.D., Lenz, O., Jeuken, L.J.C. (2014) Enhanced Oxygen-Tolerance of the Full Heterotrimeric Membrane-Bound [NiFe]-Hydrogenase of Ralstonia eutropha, J. Am. Chem. Soc., 136, 8512-8515. DOI: 10.1021/ja503138p Spotlight:DOI: 10.1021/ja5060466
In this PhD project, we aim to improve our understanding how bacteria can directly interface with electrodes via their cell membrane such that they can use the electrode as a terminal electron acceptor for their respiration. By understanding this interface we will gain insight into the bioenergetics of living cells and it will provide technolgical advances in microbial electrochemical applications.
Our initial objectives were:
1) To optimise conditions under which spheroplasts of Gram-negative and Gram-positive bacteria can bind to electrode surfaces without compromising the integrity of the cytoplasmic membrane.
2) To establish to which extend these spheroplasts are still metabolically active and are in this limited sense of the work, alive.
In the initial objectives, we aimed to electrochemically interact with the quinone pool in the plasma membran. We previously showed that this will allow us to directly control the respiratory complexes in the membrane.(2) We showed that surface-immobilised spheroplast were stable and stayed intact, as tested by using an E. coli strain that expresses a green-fluorescent protein (GFP). However, it aspired that when we used well-established biochemical methods to remove the cell-wall and prepare spheroplasts from Gram negative bacteria, large fragments of the outer membrane remain attached to the spheroplast. This tendency of the outer membrane was also described in literature of the 1960ies and 70ies, but here the outer membrane fragments prohibited the direct attachedment of the inner membrane to electrode surfaces such that the quinone pool in the inner membrane could be electrochemically controlled.
Currently, this projects has adjusted its aims and is establishing how Shewanella oneidensis MR-1, a well-studied model organism for microbial electrochemistry applications, is able to transfer terminal electrons extracellularly, for intance to electrodes they adsorbed on. We are studying the respiration of S. oneindensis MR-1 both the biochemistry or molecular level and the whole organism (microbial) level.
This project is still ongoing as the PhD programmes of Joe and Theo extend beyond the lifetime of MEME>
So far, the results of Joe and Theo have been published in the following publications:
Laftsoglou, T., Jeuken, L.J.C. (2017) Supramolecular electrode assemblies for bioelectrochemistry ChemComm, 53, 3801-3809 DOI: 10.1039/c7cc01154g
Oram, J., Jeuken, L.J.C. (2016) A re-evaluation of electron transfer mechanisms in microbial electrochemistry: Shewanella releases iron that mediates extracellular electron transfer, ChemElectroChem, 3, 829-835. DOI: 10.1002/celc.201500505
In this project, an experimental platform was developped in which membrane enzymes can be studied on the single enzyme level. In line with previously established kinetics in single-molecule enzymology, we hypothesised that cytochrome bo3 exhibit dynamic and/or static heterogeneity. This hypothesis provided an intriguing question on what happens in the presence of a thermodynamically limiting proton motive force (pmf): Does the entire enzyme population become less active (maintaining dynamic heterogeneity) or does a sub-population of enzymes become inactive (maintaining static heterogeneity).
In this project, an published methodology (3) is expanded to the single-enzyme level. Surfaces are modified sparecely with vesicles (loaded with a pH-sensitive dye), so that single vesicles can be monitored. Conditions are optimised and the presence of single vesicles are confirmed (rather than multiple aggregated vesicles). To make this approach quantitative a stable ratiometric fluorescent dye is used with simultaneous dual channel detection, to convert the fluorescent signal to pH. The enzyme content in the vesicles is reduced till single enzyme conditions are obtained (i.e., one or zero enzymes per vesicle) and the single enzyme data is analysed for static and/or dynamic heterogeneity under increasing pmf conditions.
The objectives were:
1) To optimise conditions under which vesicles loaded with fluorescent dye are adsorbed onto a gold-electrode such that single vesicles can be monitored separately using total-internal reflection fluorescence (TIRF) microscopy combined with an electrochemical setup.
2) To find an appropriate pH-sensitive dye and reconfigure the TIRF setup such that the fluorescence recordings can be used to establish the pH inside the single vesicles as a function of time.
3) To reduce the enzyme content in the vesicles such that single enzyme conditions (one enzyme per vesicle) are produced.
4) To use the single vesicle setup to determine if the proton-pumping kinetics are heterogenetic in nature (at the single enzyme level).
The project was highly succesfull and single enzyme kinetics were observed. As expected, we observed both dynamic and static heterogeneity in the activity of cytochrome bo3 and the enzymes is sometimes observed to 'stall' and tp stop proton transport even when the conditions for turn over are still present. Somewhat unexpectedly, however, we also observed rare long-lived 'leak' states during which protons rapidly leak back across the membrane. As this leak state was not observed in proteoliposomes with a mutant of cytochrome bo3, we speculate the leak state is adapted by the enzyme itself. Importantly, both the stall and leak states are dependent on the proton gradient that is formed across the membrane. Dr. Mengqiu Li now works as a reserach fellow in Oxford. This work is now published in (other publications are forthcoming):
Li, M., Khan, S., Rong. H., Tuma, R., Hatzakis, N.S., Jeuken, L.J.C. (2017) Effects of membrane curvature and pH on proton pumping activity of single cytochrome bo3 enzymes, BBA - Bioenergetics, 1858, 763-770. DIO:10.1016/j.bbabio.2017.06.003
Li, M., Jørgensen, S.K., McMillan, D.G.G., Krzemiński, L., Daskalakis, N.N., Partanen, R.H., Tutkus, M., Tuma, R., Stamou, D., Hatzakis, N.S., Jeuken, L.J.C. (2015) Single enzyme experiments reveal a long-lifetime proton leak state in a heme-copper oxidase, J. Am. Chem. Soc., 137, 6055-16063. DOI: 10.1021/jacs.5b08798
Other projects have been performed funded by the ERC programme that have not been discussed above. These have been published in:
Beales, P.A., Khan, S., Muench, S.F. and Jeuken, L.J.C. (2017) Durable vesicles for reconstitution of membrane proteins in biotechnology, Biochem. Soc. Trans., 45, 15-26. DOI: 10.1042/BST20160019
Li, K.-M., Wilkinson, C., Kellosalo, J., Tsai, J.-Y., Kajander, T., Jeuken, L.J.C., Sun, Y.-J. and Goldman, A. (2016) Membrane pyrophosphatases from Thermotoga maritima and Vigna radiata suggest a conserved coupling mechanism, Nat. Comm., 7, Art. no. 13596. DOI: 10.1038/ncomms13596
Khan, S., Li, M., Muench, S.P., Jeuken, L.J.C., Beales, P. A. (2016) Durable Proteo-Hybrid Vesicles for the Extended Functional Lifetime of Membrane Proteins in Bionanotechnology Chem. Comm., 52, 11020-11023. DOI: 10.1039/C6CC04207D
Jeuken, L.J.C (2016) Structure and Modification of Electrode Materials for Protein Electrochemistry In: Jeuken, L.J.C. (ed.) Biophotoelectrochemistry: From bioelectrochemistry to photosynthesis, Berlin: Spinger, Chapter 2. DOI:10.1007/10_2015_5011
Ye, S., Benzb, F., Wheeler, M.C., Oram, J. Baumberg, J.J., Cespedes, O., Christenson, H.K., Coletta, P.L., Jeuken, L.J.C., Markham, A.F., Critchley, K., Evans, S.D. (2016) One-step Fabrication of Hollow-channel Gold Nanoflowers with Excellent Catalytic Performance and Large Single-particle SERS Activity, Nanoscale, 8, 14932-14942. DOI:10.1039/C6NR04045D
Wiebalck, S., Kozuch, J., Forbrig, E.,Tzschucke, C.C., Jeuken, L.J.C. and Hildebrandt P. (2016) Monitoring the Transmembrane Proton Gradient Generated by Cytochrome bo3 in Tethered Bilayer Lipid Membranes Using SEIRA Spectroscopy, J. Chem. Phys. B, 120 2249 - 2256. DOI: 10.1021/acs.jpcb.6b01435
(1) Jeuken, L. J. C.; Connell, S. D.; Henderson, P. J. F.; Gennis, R. B.; Evans, S. D.; Bushby, R. J. J. Am. Chem. Soc. 2006, 128, 1711-1716.
(2) Weiss, S. A.; Bushby, R. J.; Evans, S. D.; Henderson, P. J. F.; Jeuken, L. J. C. Biochem. J. 2009, 417, 555-560.
(3) Daskalakis, N. N.; Muller, A.; Evans, S. D.; Jeuken, L. J. C. Soft Matter 2011, 7, 49-52.
Lars J. C. Jeuken
School of Biomedical Sciences
Leeds LS2 9JT UK
+44 (0)113 - 3433829
(7.19) Garstang North