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Defining the structure of ferryl heme

Reference	BB/N015940/1
Principal Investigator / Supervisor	Professor Peter Moody
Co-Investigators / Co-Supervisors	Professor Emma Raven
Institution	University of Leicester
Department	Biochemistry
Funding type	Research
Value (£)	653,804
Status	Completed
Type	Research Grant
Start date	24/02/2017
End date	31/12/2022
Duration	70 months

Abstract

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.

Summary

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.

Impact Summary

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).

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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