Defining the structure of ferryl heme
Lead Research Organisation:
University of Leicester
Department Name: Biochemistry
Abstract
Metal-catalysed activation of oxygen is a cornerstone of biology. It is usually achieved using metal ions, typically either copper or iron. In the case of iron, there are both non-heme-containing and heme-containing enzymes which activate oxygen. This application focuses on the latter.
The mechanism of activation involves formation of the iconic Compound I and Compound II intermediates, which are used across a diverse group of catalytic heme enzymes that include all the cytochrome P450s (involved in drug metabolism), the nitric oxide synthases (involved in cell signalling), and the terminal oxidases (involved in respiration), plus the heme dioxygenases (involved in formation of NAD) and heme peroxidases (involved in peroxide detoxification). Compounds I and II are such crucial intermediates in so many processes that defining their structure and understanding their reactivity, and in particular their protonation states, has been a key question in the field over several decades.
We intend to use neutron crystallography to decisively advance the field in this area. We intend to obtain neutron crystal structures of Compound II, and we propose three strategies to deliver this. This will identify the locations of all the protons in the Compound II intermediate and will allow us to make mechanistic comparisons across the family of heme enzymes.
These are ambitious, technically difficult and time-consuming experiments. But the likely gains are very high, because we have chance to finally resolve questions that have eluded the whole community for many years.
The mechanism of activation involves formation of the iconic Compound I and Compound II intermediates, which are used across a diverse group of catalytic heme enzymes that include all the cytochrome P450s (involved in drug metabolism), the nitric oxide synthases (involved in cell signalling), and the terminal oxidases (involved in respiration), plus the heme dioxygenases (involved in formation of NAD) and heme peroxidases (involved in peroxide detoxification). Compounds I and II are such crucial intermediates in so many processes that defining their structure and understanding their reactivity, and in particular their protonation states, has been a key question in the field over several decades.
We intend to use neutron crystallography to decisively advance the field in this area. We intend to obtain neutron crystal structures of Compound II, and we propose three strategies to deliver this. This will identify the locations of all the protons in the Compound II intermediate and will allow us to make mechanistic comparisons across the family of heme enzymes.
These are ambitious, technically difficult and time-consuming experiments. But the likely gains are very high, because we have chance to finally resolve questions that have eluded the whole community for many years.
Technical Summary
Catalytic heme enzymes are widely distributed in biology. They operate through the formation of highly oxidised forms of the heme, known as ferryl heme. A long-standing and highly contentious question in bioinorganic chemistry has been to clarify the protonation states of ferryl heme. Many previous attempts to resolve the question (using spectroscopy and X-ray crystallography), over several decades, have failed.
We were the first to demonstrate that neutron crystallography has the capability to convincingly resolve this issue. Here, we propose an ambitious and comprehensive experimental strategy to use neutron crystallography to define the protonation states of the ferryl heme in Compound II. We will seek to obtain structures in three related heme systems: cytochrome c peroxidase, ascorbate peroxidase and myoglobin.
We believe that our experiments will make a lasting contribution to the field, and that the information that will emerge on proton delivery and mechanism will form a framework for the development of ideas in the years to come.
We were the first to demonstrate that neutron crystallography has the capability to convincingly resolve this issue. Here, we propose an ambitious and comprehensive experimental strategy to use neutron crystallography to define the protonation states of the ferryl heme in Compound II. We will seek to obtain structures in three related heme systems: cytochrome c peroxidase, ascorbate peroxidase and myoglobin.
We believe that our experiments will make a lasting contribution to the field, and that the information that will emerge on proton delivery and mechanism will form a framework for the development of ideas in the years to come.
Planned Impact
WHO WILL BENEFIT FROM THE RESEARCH?
There are numerous beneficiaries.
1. The immediate existing personnel working with the PI and CI will benefit directly, through interactions with the project and the personnel hired on the project. This comes in the form of expertise exchanged between personnel, shared working habits, group meetings, shared learning, future collaborations between personnel once they have left the project etc.
2. The Departments involved, plus the University, also benefit. This comes through building new collaborations from outside, bringing new ideas, new ways of working, new skills, etc. The simple exchange of people across departments should not be under estimated: without it an organization becomes static, with no new input of ideas year after year. This
movement of personnel is a great benefit to UK science and UK plc.
3. The wider community, who benefit in terms of seeing how the work develops and it being a stimulus for other projects, providing ideas and a source of discussion that filters in and out of Leicester and elsewhere. Funding of new projects encourages a dialogue with other users/interested parties, which sparks new ideas and innovation elsewhere, and new
collaborations (e.g. with Edinburgh).
4. First destination employers, who benefit by picking up highly-skilled staff trained in the investigators' laboratories.
5. The wider biological community, in this country and abroad who will be interested in the results (through citations etc).
6. Heme enzymes (P450s, NO synthase for example) are a mainstay of pharmaceutical research, and this sector depends on fundamental, molecular level information emerging from academic groups around the world to prosecute their drug discovery campaigns. Our work thus feeds directly into UK plc and the contribution of molecular-level, fundamental studies
of this kind should not be underestimated.
HOW WILL THEY BENEFIT FROM THIS RESEARCH?
There are various routes through which this can be achieved.
Obviously, publication in open-access journals is one important way of publicizing information, plus attendance at national and international meetings, for which we have requested appropriate resource. We will be in regular contact with other stake-holders in the UK and abroad, and the PI is involved in organization of various events as on-going activities, such as
mini-symposia, conferences etc. This serves to publicise our work to the widest possible audience. We also routinely send our students and PDRA onto training workshops arranged by other organizations to provide training and to disseminate our work further. We are in the habit of sending PDRAs and students to smaller meetings which the PIs and CIs cannot attend
often giving talks at these events. We also have regular seminars and small meetings/conferences at Leicester, so that the ideas are publicized informally through these channels.
The University has a Business Development Office, for encouraging engagement with industry (the PI and CI have ongoing links in this area). See also Impact Statement (separate attachment).
There are numerous beneficiaries.
1. The immediate existing personnel working with the PI and CI will benefit directly, through interactions with the project and the personnel hired on the project. This comes in the form of expertise exchanged between personnel, shared working habits, group meetings, shared learning, future collaborations between personnel once they have left the project etc.
2. The Departments involved, plus the University, also benefit. This comes through building new collaborations from outside, bringing new ideas, new ways of working, new skills, etc. The simple exchange of people across departments should not be under estimated: without it an organization becomes static, with no new input of ideas year after year. This
movement of personnel is a great benefit to UK science and UK plc.
3. The wider community, who benefit in terms of seeing how the work develops and it being a stimulus for other projects, providing ideas and a source of discussion that filters in and out of Leicester and elsewhere. Funding of new projects encourages a dialogue with other users/interested parties, which sparks new ideas and innovation elsewhere, and new
collaborations (e.g. with Edinburgh).
4. First destination employers, who benefit by picking up highly-skilled staff trained in the investigators' laboratories.
5. The wider biological community, in this country and abroad who will be interested in the results (through citations etc).
6. Heme enzymes (P450s, NO synthase for example) are a mainstay of pharmaceutical research, and this sector depends on fundamental, molecular level information emerging from academic groups around the world to prosecute their drug discovery campaigns. Our work thus feeds directly into UK plc and the contribution of molecular-level, fundamental studies
of this kind should not be underestimated.
HOW WILL THEY BENEFIT FROM THIS RESEARCH?
There are various routes through which this can be achieved.
Obviously, publication in open-access journals is one important way of publicizing information, plus attendance at national and international meetings, for which we have requested appropriate resource. We will be in regular contact with other stake-holders in the UK and abroad, and the PI is involved in organization of various events as on-going activities, such as
mini-symposia, conferences etc. This serves to publicise our work to the widest possible audience. We also routinely send our students and PDRA onto training workshops arranged by other organizations to provide training and to disseminate our work further. We are in the habit of sending PDRAs and students to smaller meetings which the PIs and CIs cannot attend
often giving talks at these events. We also have regular seminars and small meetings/conferences at Leicester, so that the ideas are publicized informally through these channels.
The University has a Business Development Office, for encouraging engagement with industry (the PI and CI have ongoing links in this area). See also Impact Statement (separate attachment).
Publications
Kwon H
(2017)
Combining X-ray and neutron crystallography with spectroscopy.
in Acta crystallographica. Section D, Structural biology
Kwon H
(2020)
Neutron Crystallography in Structural Biology
Ashkar R
(2018)
Neutron scattering in the biological sciences: progress and prospects.
in Acta crystallographica. Section D, Structural biology
Moody PCE
(2018)
The Nature and Reactivity of Ferryl Heme in Compounds I and II.
in Accounts of chemical research
Kwon H
(2018)
The rise of neutron cryo-crystallography.
in Acta crystallographica. Section D, Structural biology
Kwon H
(2020)
Visualizing the protons in a metalloenzyme electron proton transfer pathway.
in Proceedings of the National Academy of Sciences of the United States of America
Kwon H
(2021)
XFEL Crystal Structures of Peroxidase Compound II
in Angewandte Chemie
Kwon H
(2021)
XFEL Crystal Structures of Peroxidase Compound II.
in Angewandte Chemie (International ed. in English)
Description | We have been able to see the pathway for proton transfer in ascorbate peroxidase using neutron crystallography. This has been accepted for publication in PNAS, crucially an arginine residue in the pathway is in the neutral (guanidine) form, We have also conducted preliminary experiment at the free electron laser (SACLA) in Japan demonstrating the feasibility of collecting diffraction data from cryotrapped intermediated with this method. We have also conducted preliminary experiment with micro-crystals at room temperature at Diamond Light Source using the "chip" technique, preliminary interpretation shows we photo-reduce and form ferrous-oxy derivatives in milliseconds. |
Exploitation Route | The observations contribute to the development of the techniques available to characterise the steps in enzyme reactions. |
Sectors | Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
Description | LMX Science Case |
Geographic Reach | Europe |
Policy Influence Type | Participation in a guidance/advisory committee |
Description | NSF(USA) Prospects in Neutron Scattering for the Biological Sciences. Alexandria VA USA Feb 20-22 2018 |
Geographic Reach | North America |
Policy Influence Type | Membership of a guideline committee |
Description | STFC Soft Matter & LIfe Sciences Advisory Panel |
Geographic Reach | National |
Policy Influence Type | Membership of a guideline committee |
Description | Japan Partnering Award -XFEL and dynamic heme enzymology |
Amount | £48,980 (GBP) |
Funding ID | BB/S020586/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 06/2019 |
End | 06/2023 |
Description | Royal Society Wolfson Fellowship |
Amount | £149,998 (GBP) |
Funding ID | RSWF\R3\183003 |
Organisation | The Royal Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 04/2019 |
End | 04/2024 |
Title | Raw XFEL diffraction data from compound II of Ascorbate peroxidase |
Description | raw X-ray data deposition for publication 10.1002/anie.202103010 and PDB ID 7BI1 |
Type Of Material | Database/Collection of data |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | making data publicly available will enable improvement in processing |
Title | Raw diffraction images of CCP by SF-ROX at SACLA |
Description | Diffraction images of cytochrome c peroxidase (CcP, PDB code: 7BIU). They were collected using X-ray free electron laser from SACLA at 15 keV with the serial femtosecond rotation crystallography (SF-ROX) method. The MX300-HS CCD detector was used to record images. CcP crystals belong to P212121 space group with a=50.81, b=75.54, c=106.57 Å. 10303 images were collected from 147 crystals, and 9145 images were merged at 1.06 Å resolution using CrystFEL 0.9.1 in the published result. The experiment was carried out on BL2. Images were converted to HDF5 files with measured photon energies. |
Type Of Material | Database/Collection of data |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | public availably of data will enable improvement of data processing methods |
URL | https://zenodo.org/record/4484116 |
Description | Contributions to Methods in Enzymology volume 634 2020 (Ed PCE Moody) |
Organisation | Oak Ridge National Laboratory |
Country | United States |
Sector | Public |
PI Contribution | I was Editor of Methods in Enzymology Volume 634 This chapter Chapter Eight - Dynamic nuclear polarization enhanced neutron crystallography: Amplifying hydrogen in biological crystals Pierce et al (2020) DOI:10.1016/bs.mie.2019.11.018 and invited this chapter because of the visit |
Collaborator Contribution | This covers the subject of the visit to the USA, the work is entirely that of the authors |
Impact | as above https://doi.org/10.1016/bs.mie.2019.11.018 in addition https://doi.org/10.1016/bs.mie.2019.11.020 is partailly an outcome |
Start Year | 2019 |
Description | Diamond Light Source |
Organisation | Diamond Light Source |
Country | United Kingdom |
Sector | Private |
PI Contribution | Provided material for X-ray crystallography and X-ray fluorescence measurements, conducted or helped to conduct experiments |
Collaborator Contribution | provided facilities and expert advice (including hands-on help) when requested |
Impact | Nature Communications article (2016) listed in outputs |
Start Year | 2014 |
Description | ILL |
Organisation | Lohengrin (Institut Laue-Langevin) |
Country | France |
Sector | Academic/University |
PI Contribution | Provision of samples (crystals) |
Collaborator Contribution | Neutron diffraction (crystallography) data collection facilities and support and processing |
Impact | publications as listed elsewhere and PhD for Cecilia M Casadei |
Start Year | 2008 |
Description | MLZ |
Organisation | Technical University of Munich |
Country | Germany |
Sector | Academic/University |
PI Contribution | This enables neutron crystallographic data to be collected at the BIODIFF beamline at FRM-II at TU Munich/Julich Centre. We provide the samples |
Collaborator Contribution | Provision of neutron source and macromolecular data collection facilities |
Impact | Publications 1) CM Casadei, A Gumiero, CL Metcalfe, EJ Murphy, J Basran, MG Concilio, SCM Teixeira,TE Schrader, AJ Fielding, A Ostermann, MP Blakeley, EL Raven & PCE Moody (2014) Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase. Science 345; 193-197 DOI: 10.1126/science.1254398. This paper was selected for F1000Prime, as being of exceptional significance in its field. 2).H Kwon, J Basran, CM Casadei, AJ Fielding, TE Schrader, A Ostermann, JM Devos, P Aller, MP Blakeley, PCE Moody & EL Raven (2016) Direct visualization of a Fe(IV)-OH intermediate in a heme enzyme Nature Communications 7, Article number: 13445 (2016) DOI:10.1038/ncomms13445 This paper was also selected for F1000Prime, as being of exceptional significance in its field. |
Start Year | 2014 |
Description | ORNL -LC |
Organisation | Oak Ridge National Laboratory |
Country | United States |
Sector | Public |
PI Contribution | We contributed expertise in the structure and mechanism of heme enzymes and the application of neutron crystallography to investigate these. We provided input into how the techniques of dynamic nuclear polarisation might be applied specifically to these (and other ) systems exploiting the paramagnetic properties of the iron in the heme group. |
Collaborator Contribution | ONRL are developing the technology and facilities required for dynamic nuclear polarisation in neutron protein crystallography. They provided expert and confidential briefings on the progress of this development. A beam-line scientist was allocated to me for a week to outline, discuss and plan means to develop the collaboration, I was granted access to administrators and the head of section to discuss and formulate was to support a collabortaion. They have also awarded me exploratory beam time and protein expression resources |
Impact | Beam time at ONRL has been awarded. The visit allowed me to assess the level of progress being made at ONRL on DNP and become familiar with the experimental set-up at the neutron crystallography beam lines at ONRL. Although a scheme to allow a PhD student to be funded by ONRL and registered at the University of Leicester was proposed and explored at length, the limitations imposed on funding meant this will not proceed for the moment. However, we are working on a proposal to support and place a Leicester-based PhD student in the ONRL laboratories for extended periods (e.g. 3 months at a time). This has strong support, but administrative hurdles remain. Subsequent to this visit I was invited to participate in a NSF-funded discussion workshop on the future of neutrons in biology held near Washington DC in February 2018 |
Start Year | 2017 |
Description | 2022 ESS/ILL European Neutron User meeting |
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 | User Meeting, Develop links with ESS |
Year(s) Of Engagement Activity | 2022 |
URL | https://indico.esss.lu.se/event/2809/overview |
Description | ENDIX 1 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Study participants or study members |
Results and Impact | Invited participant at 1st European Neutron Diffraction single crystal workshop. Abingdon April 24-26 |
Year(s) Of Engagement Activity | 2017 |
URL | https://www.cockcroft.ac.uk/events/ENDIX1/ |
Description | Endeavour Presentation |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | The ISIS Endeavour Programme - User Meeting Contribution to Biosciences and Health outlining the contribution of LMX (Large Molecule Single Crystallography station) in the upgrade My presentation is at https://www.isis.stfc.ac.uk/Pages/Endeavour-LMX.aspx |
Year(s) Of Engagement Activity | 2021 |
URL | https://www.isis.stfc.ac.uk/Pages/News21_EndeavourMeetings.aspx |
Description | ILL/ESS user Meeting |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Talk "The Hidden Secrets of Heme Peroxidases uncovered by Neutron Crystallography" at ILL/ESS user meeting October 2018 |
Year(s) Of Engagement Activity | 2018 |
Description | LINXS Reboot After the pandemics |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Talk given as contribution to meeting "to discuss the LINXS science and development in the past four years and the future ambitions in expanding the LINXS community." Lund Sweden. Establishing contacts with ESS. MAXIV and Lund University community |
Year(s) Of Engagement Activity | 2022 |
URL | https://www.linxs.se/events/2022/2/2/linxs-science-day-the-development-and-future-vision-of-linxs |
Description | LINXS/Leicester proposal meeting in Lund |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Study participants or study members |
Results and Impact | Vist to Lund to enable application for LINXS theme with Leicester Institute for Structural and Chemical Biology (LISCB) |
Year(s) Of Engagement Activity | 2022 |
Description | MLZ Conference Neutrons for Health |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Study participants or study members |
Results and Impact | A talk to a conference about the uses of "Neutrons for Health" sponsord by MLZ held in Bad Reichenhall, Germany |
Year(s) Of Engagement Activity | 2017 |
URL | https://webapps.frm2.tum.de/indico/event/48/timetable/#20170627.detailed |
Description | Meet Julich directors |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Policymakers/politicians |
Results and Impact | Meet with directors of Forschungszentrum Jülich (Germany) to discuss plans for next generation neutron sources for biological sciences research |
Year(s) Of Engagement Activity | 2022 |
Description | Neutrons in Structural Biology Grenoble 2017 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | A talk on neutron protein crystallography |
Year(s) Of Engagement Activity | 2017 |
URL | http://indico.ill.fr/indico/event/58 |
Description | Neutrons reveal (some of) the secrets of heme peroxidases |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Talk given at MLZ user conference 2021 "Neutrons for Life Science" |
Year(s) Of Engagement Activity | 2021 |
URL | https://indico.frm2.tum.de/event/230/contributions/2675/ |
Description | Nordic crystallography school |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Gave a talk and led a discussion on neutron crystallography as part of a Nordic region summer school held in Aarhus, Denmark |
Year(s) Of Engagement Activity | 2017 |
URL | http://max4essfun.ku.dk/courses/courses/data-collection-in-macromolecular-crystallography |
Description | Research Talk |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Invited talk for JuSTRUCT Inauguration (Julich Centre for structural Biology) |
Year(s) Of Engagement Activity | 2019 |
URL | https://www.fz-juelich.de/SharedDocs/Meldungen/ER-C/ER-C-3/EN/JuStruct.html?nn=2322574 |
Description | Research Talk |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | ESS Brightness2 South Africa on-line meeting. I described what we had been able to do with neutron sources. The meeting was to engage South African researchers in neutron work |
Year(s) Of Engagement Activity | 2021 |
URL | https://brightness.esss.se/news-and-press/thrust-mini-symposia-life-sciences-research |
Description | SER-CAT Symposium TSRI |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Keynote speaker at 14th annual SER-CAT meeting at The Scripps Research Institute, Jupiter FL USA |
Year(s) Of Engagement Activity | 2017 |
URL | http://petitinstitute.gatech.edu/ser-cat-conference |
Description | UK Neutron and Muon Science and User Meeting (NMSUM) 2022 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Neutron users meeting: Oral Presentation Title: "Neutrons to find deuterons in enzyme crystals > Heme Peroxidase mechanisms" |
Year(s) Of Engagement Activity | 2022 |
URL | https://www.isis.stfc.ac.uk/Pages/NMSUM2022.aspx |
Description | Workshop on Neutron Macromolecular Crystallography 31st European Crystallographic Meeting (ECM31) in Oviedo, Spain |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Workshop on Neutron Macromolecular Crystallography 31st European Crystallographic Meeting (ECM31) in Oviedo, Spain bought together those working on neutron protein crystallography |
Year(s) Of Engagement Activity | 2018 |