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.

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.

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).

Publications

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Moody P (2018) The Nature and Reactivity of Ferryl Heme in Compounds I and II in Accounts of Chemical Research

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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

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Kwon H (2017) Combining X-ray and neutron crystallography with spectroscopy. in Acta crystallographica. Section D, Structural biology

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Kwon H (2018) The rise of neutron cryo-crystallography. in Acta crystallographica. Section D, Structural biology

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Ashkar R (2018) Neutron scattering in the biological sciences: progress and prospects. in Acta crystallographica. Section D, Structural biology

 
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 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 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 05/2019 
End 04/2024
 
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 free electron laser
Collaborator Contribution Beam time at the free-electron laser, experimental data collection expertise, preliminary data proecessing
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.
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 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 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 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 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