The nature of the key reactive intermediates in heme catalysis
Lead Research Organisation:
University of Leicester
Department Name: Biochemistry
Abstract
The involvement of the metallic element iron in various biological systems is well known. In many cases, iron is employed in the form of a heme group, most famously in hemoglobin. The family of catalytic heme proteins is vast, and the majority of these catalytic heme enzymes react with oxygen in an activation process that generates oxidised forms of the heme. These oxidised forms of heme are called Compound I (oxidised by two electrons) and Compound II (oxidised by one electron). They are such crucial intermediates in so many catalytic heme proteins - including many involved in drug metabolism and other biological oxidations - that the elucidation of their structure has become a fundamental question for all those in the field.
Numerous attempts to establish the structure of Compound I and Compound II have, however, failed because of flaws in the methodology and the transient nature of the species. With improvements in methodology for neutron diffraction and for examination of iron-oxygen stretching frequencies for protein bands in crystallo (using on-line resonance Raman), it now becomes possible to take an alternative approach. We intend to use these new methods to establish, finally, the structures of these crucial intermediates.
Numerous attempts to establish the structure of Compound I and Compound II have, however, failed because of flaws in the methodology and the transient nature of the species. With improvements in methodology for neutron diffraction and for examination of iron-oxygen stretching frequencies for protein bands in crystallo (using on-line resonance Raman), it now becomes possible to take an alternative approach. We intend to use these new methods to establish, finally, the structures of these crucial intermediates.
Technical Summary
The family of catalytic heme enzymes is huge, and covers all the cytochrome P450s, the heme peroxidases, NO syntheses, cytochrome c oxidases and heme dioxygenases. Many are directly relevant to biotechnology applications and drug discovery programmes. They appear to employ a mechanism that is common to the entire family, and which involves formation of a highly oxidised heme species called Compound I. Compound I is formally oxidised by two electrons, one of which comes from an oxidised ferryl iron (usually represented as Fe(IV)=O) and one which comes from the porphyrin ring or from a protein amino acid; Compound II is oxidised by only one electron, and contains just the ferryl heme. A key question has been to establish the precise bond order and protonation state of the ferryl heme. This is a highly contentious and frequently debated subject. It is important because a definitive understanding of the key intermediates opens the way for harnessing the oxidative power of heme enzymes in an industrial/pharmaceutical context.
Over thirty years, all attempts to solve this problem have failed because of flaws in the technical methodology and there are some high profile but unreliable reports in the literature. A totally muddled picture emerges. We intend to clarify this. The aim of this proposal is therefore straightforwardly summarised: to establish, unambiguously, the structure and protonation states of the heme group in Compound I and Compound II. To achieve this, we will apply methods than have not been previously used. We will thus use neutron crystallography, which can detect protons unambiguously, and single crystal resonance Raman, which can confirm protonation states. This is technically demanding work, but we believe that the methodology is more robust than those previously applied, and that the work has the potential to resolve these question once and for all. It is highly timely, as this is a key question that has dominated the literature for many years.
Over thirty years, all attempts to solve this problem have failed because of flaws in the technical methodology and there are some high profile but unreliable reports in the literature. A totally muddled picture emerges. We intend to clarify this. The aim of this proposal is therefore straightforwardly summarised: to establish, unambiguously, the structure and protonation states of the heme group in Compound I and Compound II. To achieve this, we will apply methods than have not been previously used. We will thus use neutron crystallography, which can detect protons unambiguously, and single crystal resonance Raman, which can confirm protonation states. This is technically demanding work, but we believe that the methodology is more robust than those previously applied, and that the work has the potential to resolve these question once and for all. It is highly timely, as this is a key question that has dominated the literature for many years.
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 two 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. Clearly, this has an impact on 'quality of life', since all biotech/pharma is concerned with improvement in quality of life.
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 on-going links here).
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 two 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. Clearly, this has an impact on 'quality of life', since all biotech/pharma is concerned with improvement in quality of life.
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 on-going links here).
See also Impact Statement (separate attachment).
Publications
Moody PCE
(2018)
The Nature and Reactivity of Ferryl Heme in Compounds I and II.
in Accounts of chemical research
Ashkar R
(2018)
Neutron scattering in the biological sciences: progress and prospects.
in Acta crystallographica. Section D, Structural biology
Kwon H
(2018)
The rise of neutron cryo-crystallography.
in Acta crystallographica. Section D, Structural biology
Kwon H
(2017)
Combining X-ray and neutron crystallography with spectroscopy.
in Acta crystallographica. Section D, Structural biology
Nnamchi CI
(2016)
Structural and spectroscopic characterisation of a heme peroxidase from sorghum.
in Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry
Turner DD
(2018)
The role of Ala134 in controlling substrate binding and reactivity in ascorbate peroxidase.
in Journal of inorganic biochemistry
Kwon H
(2016)
Direct visualization of a Fe(IV)-OH intermediate in a heme enzyme.
in Nature communications
Casadei CM
(2014)
Heme enzymes. Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase.
in Science (New York, N.Y.)
Kwon H
(2020)
Neutron Crystallography in Structural Biology
Description | This project is to understand the enzyme that are responsible for the activation of oxygen. All multicellular life depends on Oxygen. We have shown that it is possible to collect neutron diffraction data from protein crystals at 100 K for long periods. This has allowed us to catch catalytic intermediates and determine their structures. Neutron crystallography shows the positions of hydrogen atoms, which is essential for the understanding of enzyme mechanisms. We have shown that much greater detail can be seen in this case by replacing all the hydrogen atoms with their heavy isotope (deuterium). We have shown that the Compound I intermediate of cytochrome c peroxidase has an unprotonated ferryl oxygen (i.e. is Fe=O) and (unexpectedly) that the distal histidine is protonated, necessitating a reappraisal of then enzyme's meachanism. We have also shown the Compound II intermediate ferry oxygen is protonated (i.e. is Fe-OH) in Ascorbate Peroxidase, resolving controversy over the nature of this intermediate. |
Exploitation Route | Cryo neutron crystallography offers many possibilities for the investigation and understanding of enzyme mechanisms. Our anaerobic glove box allows the cryo-trapping of oxygen-labile states in the crystal and these to be preserved in liquid nitrogen for diffraction analysis. |
Sectors | Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology Other |
URL | http://www2.le.ac.uk/offices/press/press-releases/2016/november/mysteries-of-enzyme-mechanism-revealed |
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 | 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 | SACLA - Spring 8 X-ray free electron laser |
Organisation | RIKEN |
Department | RIKEN SPring-8 Center |
Country | Japan |
Sector | Private |
PI Contribution | We provided ~150 crystals of the Compound II intermediate of Ascorbate Peroxidase cryo preserved for multiple exposures on large crystals x-ray data collection at the SACLA free electron laser. Further data collection in-person and by our collaborators enabled us to collect high resolution data and report 1.06 Å and 1.50 Å crystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX). The structures reveal differences between the two peroxidases, and are reported in Angewante Chemie https://doi.org/10.1002/anie.202103010 |
Collaborator Contribution | Beam time at the free-electron laser, experimental data collection expertise, preliminary data processing |
Impact | Initial results were presented at the European Crystallographic Meeting in Oviedo, Spain (2018), We have used the data to test and explore the use of the DIALS software (in collaboration with Dr Evan's group at Diamond Light Source) and are comparing the result to that from the preliminary processing with CrystalFEL. Further test are needed to optimise data collection protocols with this method, and we are applying for time to also collect data on CcP Compound II. Data is vailabel at https://zenodo.org/records/4484116 and https://zenodo.org/records/4484114 |
Start Year | 2018 |
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 | 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 | 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 | 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 | 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 | 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 | Talk at CCP4 study weekend 2016 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Lecture on methods and scope of neutron crystallography and single crystal spectroscpy to study redox states and hydrogen positions in enzymes, with particular reference to X-ray induced photoreduction. |
Year(s) Of Engagement Activity | 2016 |
URL | http://www.ccp4.ac.uk/events/CCP4_2016/ |
Description | Talk to Biology/Soft Matter session UK Neutron and Muon Users Meeting "Catching Hydrogens in Enzyme Intermediates using Cryo-Neutron Crystallography" (Warwick) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Findings from this grant-funded research presented at UK Neutron and Muon User Meeting, Delegates from UK and Europe, |
Year(s) Of Engagement Activity | 2016 |
URL | http://www.isis.stfc.ac.uk/news-and-events/events/2016/uk-neutron-and-muon-science-and-user-meeting-... |
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 | plenary lecture at the 5th International Symposium on Diffraction Structural Biology |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Use of neutron crystallography and cryo-trapping enzyme intermediates presented at (mostly USA and Japan) international symposium "The aim of this symposium is to report on the latest discoveries from the application of different biophysical techniques and computer simulations in structural biology. The meeting will encompass several thematic areas including drug design, enzyme mechanism and allostery, macromolecular complexes, membranes, and bioenergy." Discussions about the use of spin-polarised protons and collaboration with Oak Ridge neutron beam scientists initiated |
Year(s) Of Engagement Activity | 2016 |
URL | https://conference.sns.gov/event/2/ |