Professor Raven's Research Interests
Our research in chemical biology focuses on using protein engineering techniques to examine catalytic activity and function in heme enzymes. The Biological Chemistry Group collaborates extensively within the Centre for Chemical Biology at Leicester.
Current work is focused in 4 main areas:
Indoleamine 2,3-dioxygenase (supported by The Wellcome Trust).
The initial step in the l-kynurenine pathway – which is the major pathway of l-tryptophan metabolism in biology – involves the oxidation of l-tryptophan to N-formylkynurenine. This is an O2-dependent process and is catalysed by one of two heme dioxygenase enzymes: tryptophan 2,3-dioxygenase (TDO) or indoleamine 2,3 dioxygenase (IDO).
The mechanism of tryptophan oxidation is of direct interest from a biomedical/pharmaceutical perspective because overexpression of certain dioxygenases is known in a number of tumour cells, which makes these dioxygenases very attractive targets for pharmacological intervention.
We are interested in the mechanism and structure of human dioxygenases, and we use various methodologies (spectroscopy, kinetics, crystallography) to tackle molecular-level questions about their biological function. We have an on-going collaboration (funded by the Wellcome Trust) with the University of Edinburgh on this project.
Substrate binding and catalytic mechanism in heme peroxidases
(supported by BBSRC and The Wellcome Trust).
Heme peroxidases are an important class of iron-containing enzymes that catalyse the H2O2 -dependent oxidation of a variety of substrates. They contain an iron protoporphyrin IX group and are involved in a diverse range of biological functions, including antibacterial action and H2O2 detoxification.
The catalytic mechanism involves formation of a high-valent, ferryl heme intermediate followed by reduction of the oxidised heme by substrate.
The substrate specificity of these enzymes is very diverse and, although most peroxidases use small organic substrates,
the chemical determinants that define the enzyme–substrate interaction are largely unknown.
Figure 1 shows the overlay of the structures ascorbate peroxidase (red) and cytochrome c peroxidase (green). The tyrosine residue of CcP overlays with the ascorbate group in APX, blocking the binding site. The binding site for cytochrome c binding is also shown.
We have recently made major progress in understanding substrate specificity in the heme peroxidases (collaborative work with Dr Peter Moody, Biochemistry).
First, we have determined the crystal structure of ascorbate
peroxidase in complex with its physiological substrate, ascorbate (Figure 1).1
This has revealed the location of the ascorbate binding site for the first time but has also made it possible to account for the different substrate specificities observed in other heme peroxidases ( e.g. cytochrome c peroxidase). We have also identified the binding interaction of ascorbate peroxidase with other, non-physiological substrates – these bind at a different location.
Figure 2 shows the structure of ascorbate peroxidate in complex with the aromatic substrate analogue salicylhydroxamic acid.2
With BBSRC project grant and studentship support, we are now investigating how protein structure helps to define substrate specificity in both ascorbate peroxidase and cytochrome c peroxidase.
Covalent heme formation in peroxidases (supported by The Wellcome Trust).
Heme peroxidases from mammalian sources (e.g. human) are distinguished from their plant and fungal counterparts by virtue of a covalently bound heme group. Figure 3 asks 'What is the mechanism of these covalent links in mammalian heme peroxideses?' This is instead of the more usual iron protoporphyrin IX group.
With funding from The Wellcome Trust, we are interested in examining how formation of these covalent links occurs. To do this, we use HPLC and mass spectrometry together with protein engineering, kinetics and X-ray crystallography. We have recently shown that autocatalytic formation of covalently-linked heme is possible in ascorbate peroxidase by placement of a methionine residue close to the heme.3 We are currently examining the mechanism of formation of this link.
We have also shown that radical formation at other positions in ascorbate peroxidase can, under certain conditions, lead to the formation of covalent heme-protein links through mechanisms that are analogous to those used in related heme enzymes.4 Collectively, this suggests that the strategic positioning of a suitable residue within a catalytically competent protein architecture is sufficient for heme cross-linking to occur. This provides a new slant on the way we think about these covalently-modified enzymes because it suggests that some proteins may need to be posed in an environment that specifically 'switches off' these radical processes.
The experimental approach adopted in our laboratory requires a multidisciplinary approach that includes chemical, biochemical, mechanistic, redox, spectroscopic and structural techniques. Much of this work is done within the Raven laboratory and with other groups at Leicester, for example with Dr Peter Moody, Biochemistry and with the Protein and Nucleic Acid Chemistry Laboratory, but we also collaborate within the UK and abroad with structural biologists, spectroscopists and other protein chemists. As a member of the Raven group, you can expect to be exposed to a number of different chemical, biochemical and molecular biological techniques and to interact with other scientists from other disciplines at a local, national and international level.