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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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

 
Description We have been able to see the pathway for proton transfer in ascorbate peroxidase using neutron crystallography
Exploitation Route too early to say
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 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