How does respiratory complex I pumps protons? Finding the missing link using EPR spectroscopy.
Lead Research Organisation:
Queen Mary University of London
Department Name: Sch of Biological and Chemical Sciences
Abstract
The rising age of the UK population presents one of the major challenges to our society and economy and it is vital that we maximise the contribution of the most experienced individuals by increasing the number of years we live in best possible health. The mitochondrial respiratory chain, which is essential for the production of ATP and a prime source of reactive oxygen species, is thought to have a major influence on the rate at which we age. Moreover, defects in the respiratory chain are responsible for mitochondrial diseases - these are rare, but their outcome is often very severe, if not fatal. Their diagnosis is fraught with difficulties and there is no cure.
Complex I is a crucial but poorly understood element in the mitochondrial respiratory chain and we propose to elucidate a particularly important aspect of the mechanism of this essential enzyme by employing a multidisciplinary approach. We will study the bovine enzyme, a close relative of human complex I, employing in particular electron paramagnetic resonance (EPR) spectroscopy, a powerful method for investigating chemical centres having unpaired electrons. In complex I such unpaired electrons are naturally present or can be generated in mechanistically highly informative locations and we will harness the information they provide through advanced pulse EPR and biochemical methods.
Our work will constitute a major step forward in obtaining a complete picture of the molecular mechanism of one of the most important, largest and most enigmatic enzymes in our bodies, with long-term implications for increasing our healthy lifespan and for the recognition and treatment of mitochondrial diseases resulting from complex I dysfunction. Our proposed research program will have immediate impact on UK science, with academic beneficiaries in a diverse range of research disciplines. We will provide top-quality interdisciplinary training for the EPSRC PDRA (and at least two Queen Mary University undergraduate research project students), to provide expertise at the interface of chemistry and biology, with a quantitative approach to fundamental biochemical problems.
Complex I is a crucial but poorly understood element in the mitochondrial respiratory chain and we propose to elucidate a particularly important aspect of the mechanism of this essential enzyme by employing a multidisciplinary approach. We will study the bovine enzyme, a close relative of human complex I, employing in particular electron paramagnetic resonance (EPR) spectroscopy, a powerful method for investigating chemical centres having unpaired electrons. In complex I such unpaired electrons are naturally present or can be generated in mechanistically highly informative locations and we will harness the information they provide through advanced pulse EPR and biochemical methods.
Our work will constitute a major step forward in obtaining a complete picture of the molecular mechanism of one of the most important, largest and most enigmatic enzymes in our bodies, with long-term implications for increasing our healthy lifespan and for the recognition and treatment of mitochondrial diseases resulting from complex I dysfunction. Our proposed research program will have immediate impact on UK science, with academic beneficiaries in a diverse range of research disciplines. We will provide top-quality interdisciplinary training for the EPSRC PDRA (and at least two Queen Mary University undergraduate research project students), to provide expertise at the interface of chemistry and biology, with a quantitative approach to fundamental biochemical problems.
Planned Impact
We propose to elucidate the mechanism of a fundamentally important respiratory-chain enzyme, complex I, using a highly interdisciplinary approach involving both advanced spectroscopic techniques and biochemical methods. Our research is at the interface of chemistry and biology, and has the potential to generate a number of different types of impact, a summary of which is given below. Please see 'Pathways to Impact' for further details.
1) Societal Impact
As a major contributor of reactive oxygen species, complex I dysfunction has been implicated in ageing. Moreover, complex I has been shown to be the most common site for mitochondrial anomalies, accounting for as much as one third of respiratory chain deficiencies. Leber's hereditary optic neuropathy, MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and Leigh's Syndrome are some of the mitochondrial disorders specifically associated with complex I. They form part of progressive neuro-degenerative disorders, often present at birth or with onset in early childhood, for which there is no cure. Treatments are largely based on trial and error and at best alleviate symptoms. Recently, aberration of complex I activity has also been shown to enhance the progressiveness of human breast cancer cells and to result in severe hypertrophic cardiomyopathy (a disease in which the heart muscle becomes thickened). Our work in understanding how complex I functions at the molecular level will ultimately lead to rational treatment design and development and thus contribute to a better quality of life and healthy ageing.
The immediate societal impact of the research proposed will be a highly skilled researcher (the PDRA) capable of carrying out top-level research at the biology-chemistry interface. To maximise the impact of our work, we will also engage in outreach activities aimed at inspiring both the general public and the next generation of scientists for research in this field.
2) Economic Impact
As stated in the 2014 governmental budget report "funding pensions and healthcare for an ageing population will create significant cost pressures for the UK as in other countries". Increasing our 'healthspan' (the length of the healthy and active period in our lives) is thus an issue of fundamental importance to our economy and society to which our research will contribute at the fundamental level.
Another burden on our health system is the diagnosis of mitochondrial diseases, a difficult task because symptoms are often similar to a number of better-known diseases. Besides imaging, muscle biopsies have been the most successful in diagnosing mitochondrial disorders, but this invasive method is expensive and still not absolute. Treatment is often ineffective and patients usually have to be closely monitored. Development of effective treatments based on an understanding of the origins of respiratory chain deficiencies will inevitably lead to better and more cost-effective public health services and more tailored advice from charities supporting patients with mitochondrial diseases. In the absence of a clear understanding how the enzyme functions just under 'normal' conditions - the question we seek to answer - effective diagnosis and treatment is like searching for a needle in a haystack.
3) Scientific Impact
In addition to our contributions to understanding the mechanism of complex I, our work will indirectly lead to the development of applications based on proton-coupled electron-transfer reactions. Examples include the development of new catalysts capable of activating oxygen (e.g. for efficient O2 reduction to water in fuel cells) and of splitting water (one avenue to sustainable energy production). Moreover, our work will further the development of electron paramagnetic resonance spectroscopy as an extremely versatile analytical tool in applications ranging from quantum computing to understanding and exploiting processes in biology.
1) Societal Impact
As a major contributor of reactive oxygen species, complex I dysfunction has been implicated in ageing. Moreover, complex I has been shown to be the most common site for mitochondrial anomalies, accounting for as much as one third of respiratory chain deficiencies. Leber's hereditary optic neuropathy, MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and Leigh's Syndrome are some of the mitochondrial disorders specifically associated with complex I. They form part of progressive neuro-degenerative disorders, often present at birth or with onset in early childhood, for which there is no cure. Treatments are largely based on trial and error and at best alleviate symptoms. Recently, aberration of complex I activity has also been shown to enhance the progressiveness of human breast cancer cells and to result in severe hypertrophic cardiomyopathy (a disease in which the heart muscle becomes thickened). Our work in understanding how complex I functions at the molecular level will ultimately lead to rational treatment design and development and thus contribute to a better quality of life and healthy ageing.
The immediate societal impact of the research proposed will be a highly skilled researcher (the PDRA) capable of carrying out top-level research at the biology-chemistry interface. To maximise the impact of our work, we will also engage in outreach activities aimed at inspiring both the general public and the next generation of scientists for research in this field.
2) Economic Impact
As stated in the 2014 governmental budget report "funding pensions and healthcare for an ageing population will create significant cost pressures for the UK as in other countries". Increasing our 'healthspan' (the length of the healthy and active period in our lives) is thus an issue of fundamental importance to our economy and society to which our research will contribute at the fundamental level.
Another burden on our health system is the diagnosis of mitochondrial diseases, a difficult task because symptoms are often similar to a number of better-known diseases. Besides imaging, muscle biopsies have been the most successful in diagnosing mitochondrial disorders, but this invasive method is expensive and still not absolute. Treatment is often ineffective and patients usually have to be closely monitored. Development of effective treatments based on an understanding of the origins of respiratory chain deficiencies will inevitably lead to better and more cost-effective public health services and more tailored advice from charities supporting patients with mitochondrial diseases. In the absence of a clear understanding how the enzyme functions just under 'normal' conditions - the question we seek to answer - effective diagnosis and treatment is like searching for a needle in a haystack.
3) Scientific Impact
In addition to our contributions to understanding the mechanism of complex I, our work will indirectly lead to the development of applications based on proton-coupled electron-transfer reactions. Examples include the development of new catalysts capable of activating oxygen (e.g. for efficient O2 reduction to water in fuel cells) and of splitting water (one avenue to sustainable energy production). Moreover, our work will further the development of electron paramagnetic resonance spectroscopy as an extremely versatile analytical tool in applications ranging from quantum computing to understanding and exploiting processes in biology.
Organisations
- Queen Mary University of London (Lead Research Organisation)
- QUEEN MARY UNIVERSITY OF LONDON (Collaboration)
- Technical University of Munich (Collaboration)
- UNIVERSITY OF YORK (Collaboration)
- Medical Research Council (MRC) (Collaboration)
- Delft University of Technology (TU Delft) (Collaboration)
- Medical Research Council (Project Partner)
- Delft University of Technology (Project Partner)
People |
ORCID iD |
Maxie Roessler (Principal Investigator) |
Publications
Adamson H
(2017)
Retuning the Catalytic Bias and Overpotential of a [NiFe]-Hydrogenase via a Single Amino Acid Exchange at the Electron Entry/Exit Site.
in Journal of the American Chemical Society
Flanagan LA
(2016)
Re-engineering a NiFe hydrogenase to increase the H2 production bias while maintaining native levels of O2 tolerance.
in Chemical communications (Cambridge, England)
Hameedi MA
(2021)
A conserved arginine residue is critical for stabilizing the N2 FeS cluster in mitochondrial complex I.
in The Journal of biological chemistry
Hirst J
(2016)
Energy conversion, redox catalysis and generation of reactive oxygen species by respiratory complex I.
in Biochimica et biophysica acta
Le Breton N
(2017)
Using Hyperfine Electron Paramagnetic Resonance Spectroscopy to Define the Proton-Coupled Electron Transfer Reaction at Fe-S Cluster N2 in Respiratory Complex I.
in Journal of the American Chemical Society
Roessler MM
(2018)
Principles and applications of EPR spectroscopy in the chemical sciences.
in Chemical Society reviews
Wright JJ
(2016)
Small-volume potentiometric titrations: EPR investigations of Fe-S cluster N2 in mitochondrial complex I.
in Journal of inorganic biochemistry
Description | We have developed a new method that will enable a wide range of proteins to be investigated with EPR (electron paramagnetic resonance) spectroscopy. EPR-based redox (reduction-oxidation) titrations have been carried out for decades as they provide much important mechanistic information about the proteins investigated. However the limiting factor is often the amount of sample required - making traditional redox titrations impossible for the study of some proteins that are difficult to obtain in large quantities. Our method has led to a number of new collaborations and further scientific output in related disciplines (e.g. with Dr. Alison Parkin, York, resulting in two publications - see publications section). We have applied our method in conjunction with advanced EPR spectroscopy to investigate the local environment of the iron-sulfur cluster N2 in mitochondrial complex I that has been suggested to play a role in the unknown energy coupling mechanism of this enzyme or the past 30 years. We have been able to show, unambiguously, that cluster N2 is NOT directly involved in the energy coupling mechanism (Le Breton 2017, JACS, spotlight article - see publications). We have since also shown that catalysis without cluster N2 is not possible (Hameedi et al, in press in JBC, doi.org/10.1016/j.jbc.2021.100474). Our primary goal in this grant was to identify which centre, N2 or the site of quinone reduction, is involved in the energy-coupling mechanism and we have thus attained this goal. Our current efforts, beyond the scope of this grant, have been focused on showing HOW quinone binding/dissociation is involved in the coupling mechanism. To this effect, we have demonstrated that the majority of the semiquinone radical observed in submitochondrial particles (inverted membrane vesicles) is in fact originating from complex III and that it is not possible to attribute any semiquinone to complex I unambiguously (J. Wright, J. Fedor, J. Hirst and M. M. Roessler, BMC Biology, 2020). We are now working on optimising the reconstitution of complex I into liposomes for EPR studies in order to isolate any semiquinones originating from complex I unambiguously. |
Exploitation Route | The impact at this stage is on the academic sector. My group has successfully collaborated with a research group at York University (Dr Alison Parkin) in order to apply our method to study a different set of enzymes (hydrogenases) - (Flanagan et al, Chem. Commun. 2016 and Adamson et. al. J. Am. Chem. Soc 2017). Our work has also led to new projects with Dr. Guy Hanke (Queen Mary University, two joint PhD students and competitive seed funding). I have also been able to secure a Leverhulme Trust Grant which builds on some of principles developed as part of this EPSRC grant. |
Sectors | Energy,Healthcare,Pharmaceuticals and Medical Biotechnology |
Description | The main impact from this grant has been the training of a highly skilled researcher, who as a result of this training has been able to secure a permanent position in a research facility in France (as part of the Centre National de Recherche, CNRS). The facility enables the investigations of materials with unpaired electrons using electron paramagnetic resonance spectroscopy. The skills that the researcher learned during her Postdoc enable her to now train new users of the facility herself, as well as to perform a wide set of measurements that are important not only in academia but also in industry and governmental organisations. The fields that ultimately benefit from these measurements are very diverse, ranging from the investigation of new chemicals, to their environmental impact and implications in healthcare, as well as energy applications. |
First Year Of Impact | 2018 |
Sector | Chemicals,Education,Energy,Environment,Healthcare |
Impact Types | Societal,Policy & public services |
Description | Film-electrochemical EPR: a new method to investigate redox-based catalysis |
Amount | £274,302 (GBP) |
Funding ID | RPG-2018-183 |
Organisation | The Leverhulme Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 04/2019 |
End | 03/2022 |
Description | Hyperfine EPR spectroscopic investigations of the semiquinone intermediate in respiratory complex I |
Amount | £20,000 (GBP) |
Funding ID | RGS\R1\191215 |
Organisation | The Royal Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2019 |
End | 03/2020 |
Description | Life Sciences Institute Small Grant (Queen Mary University of London & Wellcome Trust) |
Amount | £23,254 (GBP) |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2016 |
End | 08/2016 |
Description | PEPR - A centre for Pulse Electron Paramagnetic Resonance spectroscopy at Imperial College |
Amount | £2,288,048 (GBP) |
Funding ID | EP/T031425/1 |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2020 |
End | 08/2024 |
Description | Principal's PhD studentship (Queen Mary University of London) - together with Dr. Guy Hanke |
Amount | £62,439 (GBP) |
Organisation | Queen Mary University of London |
Sector | Academic/University |
Country | United Kingdom |
Start | 09/2018 |
End | 09/2021 |
Description | QMUL/GCRF seed fund |
Amount | £25,694 (GBP) |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2016 |
End | 02/2017 |
Title | Small volume potentiometric titrations monitored by EPR spectroscopy |
Description | Our method is an extension of EPR-based potentiometric titrations that are widely used. The main advantage of our method is that it uses more than an order of magnitude less protein, but there are also other advantages (e.g. the possibility of EPR measurements at multiple frequencies). |
Type Of Material | Biological samples |
Year Produced | 2016 |
Provided To Others? | Yes |
Impact | In addition to our original publication (Wright et al. 2016, Journal of Inorganic Biochemistry), 3 further publications have already resulted directly from this method (Flanagan et al. 2016, Chemical Communications; Adamson et al. 2017, Journal of the American Chemistry Society; Le Breton et al. 2017, Journal of the American Chemical Society). |
Description | Collaboration with Alison Parkin |
Organisation | University of York |
Department | Department of Chemistry |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We contribute our expertise in a method developed as part of this grant (EPR-monitored small-volume redox titrations, as published in Wright et al, Journal of Inorganic Biochemistry, 2016). |
Collaborator Contribution | The Parkin group has provided us with protein samples (native hydrogenase and mutants), on which we have carried out the titrations (using our new method) and obtained EPR spectroscopic measurements. |
Impact | Two publications so far: Flanagan et al, Chem. Commun. 2016 and Adamson et al, J. Am. Chem. Soc, 2017 (please see publications section). This is a multidisciplinary collaboration involving EPR spectroscopy (Roessler group), molecular biology (Parkin group) and novel state-of-the-art electrochemical techniques (Fourier Transform alternating current Voltammetry, Parkin group). |
Start Year | 2016 |
Description | Collaboration with Dr Judy Hirst |
Organisation | Medical Research Council (MRC) |
Department | MRC Mitochondrial Biology Unit |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We are working in collaboration with the Hirst group in order to contribute to the elucidation of the mechanism of mitochondrial complex I through EPR spectroscopic methods. |
Collaborator Contribution | The Hirst group has contributed their extensive experience in working with bovine complex I, training members of my group to carry out the purification and thus enabling us to carry out the purification in house. We have also had access to unpublished work and benefited from the intellectual input to our publication. |
Impact | Judy Hirst and I have co-authored one review paper and another primary research article is submitted. |
Start Year | 2015 |
Description | Collaboration with Guy Hanke |
Organisation | Queen Mary University of London |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | In this project with Guy Hanke we are investigating the mechanism of the so-called NDH complex in plants. This is an enzyme which is highly homologous to respiratory complex I, which we have been studying extensively as part of this EPSRC grant. There are currently no biophysical studies of the NDH complex at all, and we are contributing our expertise on complex I to this project - both in terms what we know about its mechanisms, but also in terms of biochemical and biophysical tools to study these large molecular machines effectively. |
Collaborator Contribution | Dr Guy Hanke has the expertise to isolate the NDH complex from different species (cyanobacteria and higher plants) and to genetically modify it as required for detailed mechanistic investigations. |
Impact | In preliminary work we have already isolated the enzyme NDH from cyanobacteria (work carried out by a PDRA hired on EPRSC-GCRF seed funding, see 'further funding' section) and obtained first EPR spectroscopic measurements on this enzyme. This enabled us to secured a competitive PhD studentship on this project and we are also in the process of applying for RCUK funding. |
Start Year | 2016 |
Description | Collaboration with Prof. de Vries |
Organisation | Delft University of Technology (TU Delft) |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | Unfortunately Prof. Simon de Vries unexpectedly passed away in September 2015. Prof. de Vries was a named collaborator on the grant to enable us to carry out freeze quench investigations in the latter part of the project. He was due to visit Queen Mary University in January 2016 to give a seminar and to discuss detailed experiments. I am now in touch with Prof. Wilfred Hagen (TU Delft) to see how we may be able to carry out the freeze-quench experiments. |
Collaborator Contribution | Unfortunately Prof. de Vries could not make a contribution since the project started less than 2 months before he passed away. |
Impact | N/A (please see above) |
Start Year | 2015 |
Description | Collaboration with Ville Kaila |
Organisation | Technical University of Munich |
Country | Germany |
Sector | Academic/University |
PI Contribution | We contribute our expertise in carrying out advanced EPR spectroscopic measurements and analysis of respiratory complex I to this collaboration. |
Collaborator Contribution | The Kaila group is a leading expert in theoretical/computational investigations of respiratory complex I. They are able to generate experimentally-testable predictions that save much experimental effort (time and money). |
Impact | This is a new collaboration that resulted from Prof. Ville Kaila and myself meeting at the Bioenergetics Gordon Research Conference in June 2017. At the moment, we are missing the dedicated manpower to advance the project, but a student from Turkey (Esra Korpe) is due to start her PhD in September 2018 (see 'Further Funding' Section) and although Esra will be primarily based at QMUL she will spend research periods in the Kaila group. The collaboration is multidisciplinary, involving biophysics, biochemistry and advanced computational methods. |
Start Year | 2017 |
Description | Freeze-quench studies of respiratory complex I |
Organisation | Delft University of Technology (TU Delft) |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | This is a new collaboration with Prof. Peter-Leon Hagedoorn following the unexpected death of Prof. Simon de Vries. Myself and a member of my research team visited Delft in May 2017 with a first set of enzyme (complex I) samples. Our expertise (accumulated over the course of this grant) therefore consisted of preparing the high-quality samples required in order to carry out the freeze-quench experiments. |
Collaborator Contribution | Delft has a world-wide unique freeze-quench instrument that we have free-of-charge access to during our visit. The operation of the instrument is highly skilled and was carried out by the resident technician who was involved in building the instrument together with Prof. de Vries. In addition, continuous-wave EPR experiments of the freeze-quench samples were carried out by Prof. Wilfred Hagen. Although we do have the expertise to carry out EPR measurements at Queen Mary University of London, on-site measurements of the newly-prepared freeze-quench samples was required to assess the outcome of each freeze-quench experiment and to plan subsequent freeze-quench experiments effectively. In addition to instrument access, Prof. Hagedoorn and his team provided important intellectual input into the design of the freeze-quench experiments based on their expertise in this area. |
Impact | We successfully completed first freeze-quench and EPR experiments of bovine complex I in Delft and this data will serve as preliminary data for a future grant application. This is a multi-disciplinary collaboration: Freeze-quench (Delft) and biological EPR spectroscopy (QMUL). |
Start Year | 2017 |
Description | As STEM ambassador contributed to QMUL Centre of the Cell 'Pod Shows' (a short talk about my work to secondary school children) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | The 'Pod' is part of the 'Centre of the Cell' at QMUL. The Pod provides a learning experience of school children and enables them to get a flavour for scientific research. School children are able to try their hand at scientific experiments during the Pod show. Prior to each pod show, an academic volunteer gives a brief talk (ca. 10 minutes) about their work and children are given the opportunity to ask questions - this was my role as a STEM ambassador during this event. Approximately 30 students and teachers attending this pod show and my short talk received (in which I explained in lay terms what my research on complex I involves) many questions from both pupils and sparked further discussion with teaching during and after the show. |
Year(s) Of Engagement Activity | 2016 |
URL | https://www.centreofthecell.org/ |
Description | Host of the 51st Annual International Meeting of the Electron Spin Resonance Group of the Royal Society of Chemistry |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | This is the most important EPR conference in the UK, that attracts national and international participants as well as extensive interest from industry (who is generously sponsoring the conference). I was asked to host the conference at QMUL not least due to the output generated from this EPRSC grant that demonstrates the rising impact of the EPR facility at QMUL. |
Year(s) Of Engagement Activity | 2018 |