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.

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.

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).

Publications

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

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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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Moody PCE (2018) The Nature and Reactivity of Ferryl Heme in Compounds I and II. in Accounts of chemical research

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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

 
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 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 Diamond Light Source 
Organisation Diamond Light Source
Country United Kingdom 
Sector Academic/University 
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 Public 
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 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 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 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 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/