The nature of the key reactive intermediates in heme catalysis

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.

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.

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).