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|Title:||An investigation of heme-protein covalent links and active site cross-links in heme peroxidases|
|Presented at:||University of Leicester|
|Abstract:||Proteins and enzymes that contain a heme group form a diverse family that are involved in a truly overwhelming range of biological processes. Many heme proteins contain iron protoporphyrin IX, however, it is now becoming clear that a large number of other proteins, e.g. the mammalian peroxidases, use modified versions of iron protoporphyrin IX in which the heme is covalently linked to the protein. This thesis presents an investigation of covalent heme attachment in APX and plant proteins.;In Chapter 2, it was shown that reaction of APX with H2O 2 leads to the formation of a covalent link from the heme to Trp41 residue under non-catalytic conditions. Formation of this covalent link was proposed to proceed through a Compound I species bearing a porphyrin pi-cation radical. Formation of a protein radical at Trp41 is also implicated, in a reaction mechanism that is analogous to that proposed for formation of a covalent Trp-Tyr-Met link in the closely related catalase-peroxidase (KatG) enzymes. It was also shown that the same covalent link is formed into the S207E variant of APX.;In Chapter 3, it was shown that a covalent link between the heme and Trp51 cannot be supported by wild type CcP, but can be engineered into the W191F variant of CcP where formation o f a Compound I species bearing a porphyrin 7c-cation radical is sustainable. A comparison o f the similarities and differences between the mechanisms used by the members o f the Class I family o f plant peroxidases is made in both Chapters 2 and 3.;In Chapter 4, the reaction o f the S160Y variant o f APX with H2O2 was examined and it was shown that Tyrl60 forms a covalent link to the heme in an autocatalytic reaction that also leads to formation o f a second covalent link to Trp41, as above. The formation o f these links was found to have a profound effect on the redox properties o f the heme iron. The implications o f these data are discussed in terms of both current understanding o f heme group reactivity and the conditions needed for any heme protein to duplicate the active site architecture observed in the mammalian peroxidases.;Although several questions still remain unanswered, the work in this thesis has given valuable insight into the formation o f covalent links in heme proteins, using plant proteins as model systems and has demonstrated how plant proteins can provide alternative routes for studying covalent heme attachment.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Chemistry|
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