Cytochrome c oxidase: structure, function and malfunction

Lead Research Organisation: Birkbeck College
Department Name: Biological Sciences

Abstract

To live we need a permanent supply of energy. This is provided to our cells by a cascade of reactions that breaks down the food we eat into a universal fuel: the ATP. This process mainly occurs in organelles called mitochondria and is known as cellular respiration. The main machinery that mitochondria use to produce ATP is the respiratory chain. It is composed of four complexes, embedded in the mitochondrial inner membrane, that work together to build up an electrochemical gradient called the proton motive force and which drives ATP synthesis. Most of this gradient is in the form of protons which are pumped across the inner mitochondrial membrane by the respiratory chain complexes.

An increasing number of human pathologies are associated with defects in components of the respiratory chain. In many instances, this is because the malfunction has a direct impact on their primary role in energy production via the proton gradient that they form, or because it leads to an increased production of damaging free radicals. Cytochrome c oxidase (CcO) is the terminal enzyme of our respiratory chain. It transforms the oxygen we breathe into water and greatly contributes to the generation of the proton gradient. Alterations (or mutations) in its structure have been linked with diverse pathologies such as myopathy, therapy-resistant epilepsy, neurological diseases and prostate cancer.

Although the overall chemistry of mitochondrial CcO is fairly well understood, it has proven much more difficult to determine how this produces the essential proton gradient. Various hypotheses were formulated based on the available structures of the enzyme (of which only one is of mitochondrial origin, in this case bovine), but were challenged by mutagenesis work performed on smaller bacterial homologues. Today it appears that the major drawback in understanding the mechanism of mitochondrial CcO, and the effects of human disease-related mutations in particular, is the lack of a system to generate large amounts of purified protein containing defined point mutations.

Remarkably, the CcO that is present in Baker's yeast mitochondria is almost identical to that in human mitochondria. The nuclear and mitochondrial DNAs which encode CcO are both amenable to mutagenesis so alterations can be made in any part of the CcO structure to investigate its function. We have thus engineered a yeast system to allow large-scale production of mutants and will use it to address fundamental questions relative to human mitochondrial CcOs.

At first, we will identify the route taken by the protons to cross the protein structure by measuring CcO's ability to pump protons after alterations have been made in chosen area. We will then use advanced techniques like infrared spectroscopy to look at the concerted movement of atoms within CcO's structure and bring experimental evidences of the mechanism following which protons are being pumped. This should tell us more about the principles that govern and control the complex activity and will be our starting point to investigate how factors or signals external to the reaction centre can, in vivo, regulate CcO's activity. This will be of particular interest to understand how the human CcO has adapted to different energy requirements depending on tissue type. We will aim to obtain a detailed 3D structure of the yeast CcO to confirm our hypotheses. As we unravel the details of CcO's action, we will introduce identified human disease-related mutations in our yeast system in order to investigate the nature of their malfunction. Finally, we will aim at progressively incorporating the human genes or parts of the human enzyme in our yeast system. This will create as even better model for the study of human diseases and the development and testing of new therapies.

Technical Summary

The aim of this project is to elucidate the pumping mechanism of CcO and to investigate the effect of allosteric factors and isoforms on catalysis, using, for the first time, a yeast system that allows production and large scale purification of mitochondrial mutant forms of the enzyme. We will also start developing yeast as a platform for the construction of a chimeric yeast/human CcO.

Initial focus will be put on identifying the internal hydrophilic pathway responsible for proton pumping. This will involve accurate measurements of ADP/Oxygen ratios on preparations of intact mitochondria from mutant strains.

We will then address the coupling mechanism using time-resolved FTIR spectroscopy to detect concerted movements of key amino acids and water molecules on chosen reaction steps with the purified CcO. This will be done on photolysis of the fully reduced CO-bound enzyme, comparing the signals recorded for the wild-type and mutant forms and, subsequently, be extended to redox reactions.

We will investigate how long range factors can regulate catalysis. For instance, we will follow by FTIR spectroscopy the structural changes induced by the binding of ADP or ATP to allosteric sites on yeast supernumerary subunits. This will include the design of a range of mutants to mimic post-translational modifications such as phosphorylation.

We will aim to crystallise the yeast CcO and resolve its 3D X-ray structure.

Finally, we will use our yeast system to investigate the effect of human disease-related mutations in CcO. We will use a range of biochemical methods to determine level of expression/stability, turnover number, affinity for its substrate, ability to pump proton and at which stoichiometry. If required, more advanced biophysical techniques including FTIR spectroscopy and fast kinetics recording will be used. Ultimately, as the project develops, we will look at the effect of such alterations on a chimeric yeast/human enzyme.

Planned Impact

Who will benefit from this research?
-researchers in biological electron transfer and energy coupling;
-UK and worldwide Research Centres (e.g. the Mitochondrial Biology Unit in Cambridge, The Mitochondrial Centre, Newcastle, the Mitochondrial Research & Innovation Group, Rochester, USA) and medical charities and public domain sites (e.g. The United Mitochondrial Disease Foundation) devoted to mitochondrial research and its wider implications;
-members of the EU COST Action CM1306 (Understanding Movement and Mechanism in Molecular Machines) whose main objective is to improve understanding of the dynamics of protein complexes on catalysis by bridging neighbouring scientific fields and fostering applicative outcomes;
-patients and families of patients affected by CcO-related mitochondrial diseases.

How will they benefit from this research?
Basic researchers in related areas will benefit from the mechanistic understanding and structures important for energy coupling that should find applicability in other biological redox systems. Moreover, if the behaviour of yeast mutants is as predicted, a common picture for all forms of CcO will have been established, providing a resolution of current CcO mechanism controversies.
Members of the more diverse organisations above will be particularly interested in our development of this yeast CcO system as a platform for understanding mechanism and control of human mitochondrial CcO function and its malfunction in diseases. This yeast system will provide the only currently viable genetic system to test effects of known mutations of human mitochondrial CcO, using an enzyme that closely resembles its structure in all key aspects. Its extension into a system that assembles a chimeric yeast/human enzyme will also be of great benefit for patients as it can be used for the development and testing of new therapies.

Timescales. Within the timeframe of the project, we will bring new experimental evidences of the coupling mechanism, establish the function of the H channel region of mitochondrial forms of CcO and provide insights into how such enzymes are modulated by environmental factors. This yeast system is already usable to test effects of known human CcO mutations that have been linked to disease states. We will work closely with our clinical collaborator Dr Rahman, involved in collecting such mutational/clinical data to gain a mechanistic understanding of their malfunction. During this project lifetime, and in collaboration with Dr Meunier, we will extend the technology and expertise specifically to create a chimeric yeast/human system. On a longer term, this should lead to a viable platform for the detailed characterisation of the mechanisms underlying CcO-related human pathologies.

Staff development. The PDRA involved in the project will gain expertise with a wide range of biochemical and advanced biophysical methods. Through our collaborations with other laboratories in London (Peter Rich, respiratory complexes), France (Brigitte Meunier, genetics) and Cambridge (Leo Sazanov, crystallography) they will gain additional skills. Through my participation in EU COST Action, funds will be available for exchange between laboratories and myself and the PDRA will apply for COST-funded 'Short Term Scientific Missions' to learn expertise of our collaborators and in particular with the group of Peter Brzezinski (Sweden). The research skills that will be gained during this project are applicable to other major enzymes, including several of medical interest. In addition, professional skills that staff will develop will be applicable in other employment sectors for example ability to multi-task, work in a team and communicate with scientists and non-scientists. Hence, the project will provide skills for onward employment and career development.

Publications

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Liu C (2018) Phosphonomethyl Oligonucleotides as Backbone-Modified Artificial Genetic Polymers. in Journal of the American Chemical Society

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Hartley AM (2019) Structure of yeast cytochrome c oxidase in a supercomplex with cytochrome bc. in Nature structural & molecular biology

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Maréchal A (2020) A common coupling mechanism for A-type heme-copper oxidases from bacteria to mitochondria. in Proceedings of the National Academy of Sciences of the United States of America

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Hartley AM (2020) Rcf2 revealed in cryo-EM structures of hypoxic isoforms of mature mitochondrial III-IV supercomplexes. in Proceedings of the National Academy of Sciences of the United States of America

 
Description Birkbeck / Wellcome Trust Institutional Strategic Support Fund (ISSF)
Amount £24,971 (GBP)
Organisation Birkbeck, University of London 
Sector Academic/University
Country United Kingdom
Start 04/2018 
End 10/2018
 
Description Birkbeck / Wellcome Trust Institutional Strategic Support Fund (ISSF)
Amount £22,917 (GBP)
Organisation Birkbeck, University of London 
Sector Academic/University
Country United Kingdom
Start 06/2015 
End 05/2017
 
Description Royal Society Research Grants Scheme
Amount £15,000 (GBP)
Funding ID RG150631 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2016 
End 03/2017
 
Title Deposition of 1 entry to the Protein Data Bank 6HU9 
Description This entry to the Protein Data Bank provides atomic details of the structure of yeast mitochondrial cytochrome c oxidase in a supercomplex with cytochrome bc1 in the biologically relevant 2:2 stoechiometry. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Saccharomyces cerevisiae is the ideal model system universally used to test/verify hypothesis in the study of mitochondrial respiratory chains. This model will permit the validation of past and future mutagenesis studies and will stand as a reference across disciplines for the advancement of knowledge. 
URL https://www.ebi.ac.uk/pdbe/entry/pdb/6hu9
 
Title Deposition of 3 electron microscopy maps into the Electron Microscopy Data Base EMD-0262, EMD-0268, EMD-0269 
Description These three EM maps are the result of extensive image processing which led to the structure, at atomic resolution, of the supercomplex formed by mitochondrial complexes IV and III 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Together, these maps allowed the construction of the first atomic model of a mitochondrial III-IV supercomplex (deposited in the PDB as 6HU9) 
URL https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-0262/index
 
Description Crystallisation and structure determination of cytochrome c oxidase from Saccharomyces cerevisiae 
Organisation Institute of Science and Technology Austria
Country Austria 
Sector Academic/University 
PI Contribution We send them samples of cytochrome c oxidase purified to crystallisation quality from yeast S. cerevisiae and setup crystallisation trials on the fresh preparations under their guidance.
Collaborator Contribution They guide us to improve the stability/purity of our protein preparation and perform extensive crystallisation screens on frozen samples of the enzyme.
Impact At today no output or outcome can be reported. Disciplines involved: Biochemistry, Biophysics.
Start Year 2015
 
Description Effect of mutations in cytochrome c oxidase from Saccharomyces cerevisiae 
Organisation Institute for Integrative Biology of the Cell (I2BC)
Country France 
Sector Academic/University 
PI Contribution We study and analyse usign a combination of biochemistry and biophysics methods the effect of point mutations on the catalytic activity of cytochrome c oxidase.
Collaborator Contribution They provide us with strains of yeast S. cerevisae containing mutation in their cytochrome c oxidase (both nuclear and mitochondrial-DNA encoded subunits) to test/verify hypothesis.
Impact Several publications since 2012. Disciplines involved: genetics, biochemistry, biophysics.
Start Year 2012
 
Description Effect of point mutations and isoform on the kinetics of the reaction of yeast cytochrome c oxidase with dioxygen 
Organisation Stockholm University
Department Department of Biochemistry and Biophysics
Country Sweden 
Sector Academic/University 
PI Contribution We have identified the mutant strains of interest from their level of oxidase expression, UV visible absorption signature and rate of oxygen consumption in comparison with WT. These are point mutations of I67N, N99D in subunit I and the constructs of the two natural isoforms of the yeast oxidase where only COX5A or 5B is expressed. We then grow the cells and make mitochondrial membrane preparations of the selected mutant oxidase or purify it. This material is then used for the flow-flash experiments in Stockholm.
Collaborator Contribution They have the specialist flow-flash apparatus that permits fast kinetics measurements on reaction of the four or two electron-reduced WT and mutant forms (or isoforms) of oxidase with molecular oxygen. They can also measure proton uptake by the enzyme on selected catalytic steps.
Impact Exchange of knowledge, training of PhD students and PDRA. Disciplines involved: biophysics, biochemistry, molecular biology.
Start Year 2016
 
Description Mitochondrial Techniques Workshop 12-13 September 2017, UCL, London 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Two day, laboratory based workshop focused on techniques for mitochondrial research. I led the session on using UV/visible and infrared spectroscopy to gain qualitative, quantitative and structural information from the components of the mitochondrial respiratory chain
It was held at University College London and was hosted by the UCL Consortium for Mitochondrial Research (CfMR) with support from The Physiological Society, the Biochemical Society and the British Pharmacological Society.
Year(s) Of Engagement Activity 2017
URL http://www.physoc.org/mitochondriaformandfunction/mitochondrial-techniques-workshop
 
Description Organisation of the 79th Harden Conference on 'Oxygen Evolution and Reduction - Common Principles' 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact This conference aimed at gathering the expertise of scientists working on photosystem II and cytochrome oxidases, two enzyme systems for which mechanistic and structural questions are common but which are rarely discussed comparatively. The format of the Harden meeting as a residential (comparable to a Gordon conference) fostered discussions and presentations of newly/unpublished ideas and data.
The feedback received on the meeting from all the participants was extremely positive.
Year(s) Of Engagement Activity 2016
URL https://www.biochemistry.org/Events/tabid/379/MeetingNo/79hdn/view/Conference/Default.aspx
 
Description Organisation of the 85th Harden Conference on 'Dynamic Membrane Complexes: Respiration and Transport' in Bonn, Germany 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact The programme of this residential conference was designed to bring leaders in the fields of molecular respiration together with scientists working at the forefront of membrane transport, membrane structural biology and lipid biochemistry to create a more complete understanding of respiration and transport and explore further the relationship between function and disease.
The conference has received tremendous feedback and the organisation of a next edition is already well underway for 2021.
Year(s) Of Engagement Activity 2019
URL https://biochemistry.org/events/85th-harden-conference-dynamic-membrane-complexes-respiration-and-tr...
 
Description Organisation of the Annual Meeting of the Bioenergetics group UK, Birkbeck, London 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact The 1-day Bioenergetics Christmas meeting was held at Birkbeck College on the 19th Dec 2017.
It started with a keynote lecture by Prof Bill Rutherford FRS, from Imperial College, entitled 'Photosynthetic reaction centers: relating light, oxygen, survival and the energy limits' followed by 10 short talks from Post-docs and PhD students.
Prof Rutherford gave a brilliant account of the state of the research in photosynthetic reaction centres, presenting some very exciting ideas and new perspectives to the field. He has certainly been very inspirational to the young researchers in the audience and has sparked some animated discussions among more senior scientists from which we all benefited over lunch.
The presentations that followed from post-docs and PhD students were equally exciting and all were very thankful for the opportunity to present their work to an informed audience.
The meeting was very well attended with people coming from across the country (and the world actually), the majority having last met a year or two ago at another edition of the Christmas meeting. I could hear people resurrecting plans of collaborations, other starting new ones (including myself) and there was a real sense of enthusiasm and optimism. It has been an excellent networking event for the UK bioenergetics community, with many very senior researchers present, discussing the meeting with junior colleagues.
Year(s) Of Engagement Activity 2017
 
Description Poster presentation by 1st year Wellcome Trust PhD rotation student Gabriel Ing at the annual UK Bioenergetics group meeting - Queen Mary University of London, 16 December 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Gabriel presented his work in the form of a poster. This was his first participation in a scientific meeting and gave him exposure to the national bioenergetics community.
Year(s) Of Engagement Activity 2019
 
Description Scientific presentation given by Dr Andrew Hartley at the Annual Meeting of the Bioenergetics group UK, Birkbeck, London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Dr Andrew Hartley presented his latest results in a presentation entitled 'Cryo-EM studies of cytochrome c oxidase and respiratory supercomplexes from Saccharomyces cerevisiae'.
Year(s) Of Engagement Activity 2017
 
Description Talk by Dr Andrew Hartley at the 85th Harden Conference in Bonn, Germany - 28 August 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Andrew presented his latest unpublished work on cryoEM of yeast respiratory supercomplexes.
Year(s) Of Engagement Activity 2019
URL https://www.eventsforce.net/biochemsoc/frontend/reg/tAgendaWebsite.csp?pageID=26044&ef_sel_menu=343&...
 
Description Talk by Dr Andrew Hartley at the Annual Meeting of the Bioenergetics group UK, Cambridge - 14 December 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Dr Andrew Hartley presented our work to our UK scientific community on the determination of the structure of cytochrome c oxidase by cryoEM.
People inquired in which journal they could find the details of our work which was very well received by the audience.
Year(s) Of Engagement Activity 2018
 
Description TechMuses One-day visit from students from Gladesmore Community school to UCL 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Every year, since 2018, I welcome to my lab at UCL twenty 14-15-year-old girls and their teachers from Gladesmore community school (http://www.gladesmore.com/), a less-privileged girl-only school in Haringey. I talk to them about my research and career path. The visits are in partnership with a charity called Techmuses that provide mentorship to school girls that are interested in STEM subjects.
Each year the visit is a huge success and the school is immensely grateful for providing to their pupils such an open minding insight into STEM research.
Year(s) Of Engagement Activity 2018,2019,2020
 
Description Technical workshop demonstrating the application of FTIR spectcroscopy to PhD students, postdocs and established scientists in the field of mitochondrial research 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact The course entitled 'Principles of Mitochondrial Biology, Metabolism and Bioenergetics in Health and Disease' was designed primarily for PhD students and post docs but also catered for more established academics seeking a deeper understanding of mitochondrial biology and techniques applicable to their research.
In particular, the extent of the application of FTIR spectroscopy to biological studies is not well known so its dissemination to skilled scientists, at any stage of their career, has the potential to directly benefit their field of expertise.
Advises were given to all participants on the applicability of the technology to their own research.
Year(s) Of Engagement Activity 2015
URL http://mitochondria.cs.ucl.ac.uk/mip2015/