Our research interests centre on the genetic modification of haem iron enzymes. Iron is the most widely used metal catalyst in biological systems, and haem enzymes, such as peroxidases, catalyses and monooxygenases, catalyse a number of key chemical reactions. We aim to study the role of individual amino acids in enzyme activity, and to use genetically engineered enzymes to carry out chemical transformations, e.g. C-H bond activation, which are difficult to achieve using conventional methods.
1. Modelling Haem Enzyme Activity:
The haem oxygen-storage protein myoglobin is one of the best characterised proteins. Our aim is to examine the specific features of haem proteins which promote their catalytic activities, using myoglobin as a model. We are targeting specific amino acids in the active site of myoglobin for alteration by site specific mutagenesis. Such changes affect the haem reduction potential, the size and polarity of the haem pocket, and specific interactions such as hydrogen bonding, all of which could be important in determining the catalytic activity. A single amino acid change we have made improves the catalytic activity of myoglobin, to a level comparable with naturally occurring haem enzymes.
2. Changing the Substrate Selectivity of enzymes:
We are changing the substrate selectivity of cytochrome P-450cam, a haem enzyme that catalyses the hydroxylation of C-H bonds, to prepare new biosensors and biocatalysts for chemical synthesis. Amino acids, whose side-chains provide specific contacts for the molecular recognition of substrates, are being altered by site specific mutagenesis. One mutant we have constructed binds molecules which do not bind to the naturally occurring protein. We combine single-crystal X-ray diffraction and solution NMR studies to determine protein-substrate contacts in the new proteins. This data then allows us to design further mutations to achieve higher selectivity.
Substrate binding can dramatically affect the electrochemical properties of the P-450cam active site, and the new proteins could be used as biosensors. Hydroxylation of substrate C-H bond(s) could be used to carry out important steps in chemical synthesis. This work is being carried out in collaboration with Prof H.A.O. Hill F.R.S., and Dr S.L. Flitsch.
3. The Effect of Fatty Acids on Cytochrome c:
The haem protein cytochrome c transfers electrons to cytochrome oxidase, which in turn reduces oxygen to water, in the terminal reaction of the mitochondrial respiratory chain. Excessive levels of fatty acids have been shown to inhibit this reaction, most likely by direct interaction with cytochrome c. In collaboration with colleagues in Canada and the U.S., we have examined the interaction between cytochrome c and fatty acids using computer modelling, and are studying the proposed binding site by a combination of site-specific mutagenesis, NMR spectroscopy, and X-ray diffraction. The cytochrome c/cytochrome oxidase reaction in the human heart has been shown to be affected by fatty acids, therefore the results of this study might have significant health implications.
4. Design of Ion-Selective Biosensors:
We have genetically modified the surfaces of small redox active proteins to provide selective binding sites for Group 1 and 2 metal ions. The location of these sites were chosen such that binding of cations affect, in a concentration-dependent manner, the redox potential of the protein active site. Based on our knowledge of the metal environments in coordination compounds, we have shown that it is possible to alter which metal is selectively bound, by changing the amino acid side-chains which provide the ligands to the metal ion. The new proteins could thus be used as biosensors for medically important alkali and alkaline earth metal ions. The key advantage of using a redox active protein as the sensor is that the analysis can be carried out in aqueous solution. This work is carried out in collaboration with Prof H.A.O. Hill F.R.S.