Please use this identifier to cite or link to this item:
|Title:||Human indoleamine 2,3-Dioxygenase|
|Authors:||Papadopoulou, Nektaria D.|
|Presented at:||University of Leicester|
|Abstract:||We have developed an efficient bacterial expression system for production of human IDO (hIDO) that utilizes a His-tag expression vector and have established reliable protocols for purification of the recombinant protein. We have used this expression system to examine the redox, spectroscopic and substrate-binding properties of the enzyme. The Fe3+/Fe2+ reduction potential was found to be -30 +/- 4 mV; in the presence of L-Trp, this value increases to + 16 +/- 3 mV. Electronic, EPR and MCD spectroscopies indicate that ferric rhIDO (pH 6.6) exists as a mixture of six-coordinate, high-spin, water-bound heme and a low-spin species that contains a second nitrogenous ligand. There is an increase in the low-spin component at alkaline pH for rhIDO, but this is not due to hydroxide-bound heme. Substrate binding induces a conformational rearrangement and formation of low-spin, hydroxide-bound heme.;The role of two key histidine residues, H346A and H303A, was also examined in Chapter 4. Parallel spectroscopic, electrochemical and ligand binding analyses were consistent either with H303 as the sixth ligand or with H303 linked to a conformational change that affects the formation of the low-spin heme species. The first scenario is ruled out in light of the recently published crystal structure of rhIDO. Further analyses of the H303A variant indicated that this residue is not required for the formation of the low-spin, hydroxide-bound heme in the presence of the substrate. The Fe3+/Fe2+ reduction potential of H303A variant is "70 mV lower than that of rhIDO, leading to a destabilization of the ferrous-oxy complex, which is an obligate intermediate in the catalytic process.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Chemistry|
Items in LRA are protected by copyright, with all rights reserved, unless otherwise indicated.