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Columbia Dissertations and Theses > Doctoral Dissertations
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| Author(s): | Ching, Koon-Cheung |
| Title: | Using density functional theory to probe the role of axial ligands in heme-protein thermodynamics/ |
| Physical Description: | ix, 106 leaves, bound. |
| Issue Date: | 2008 |
| Description: | Department: Chemistry. UMI cat no. 3305208 Thesis (Ph. D.)--Columbia University, 2008. |
| Bookmark as: | http://hdl.handle.net/10022/AC:P:18513 |
| Full Text (ProQuest): | /ac/proxit.jsp?url=http://gateway.proquest.com/ope... |
| Abstract: | Development of computational tools that accurately model interactions between the primary coordination sphere and the heme cofactor is essential in achieving rational design of heme proteins with specific functions. We report a protocol based on Density Functional Theory (DFT) for calculating relative binding affinities and reduction potentials of iron protoporphyrin (IX) cofactor (Fe-PPIX) bound by pairs of ligands. The following side-chain groups are examined: imidazole (His), 3-methyl-imidazole (3meHis), 1,2,4-triazole (Tzl), tetrazole (Ttr), and pyridine (Pal4). The binding affinities and reduction potentials are calculated relative to His-His ligation since this coordination motif is the most widely observed ligation scheme in biology. The accuracy of calculated values is assessed through comparison with experimental values measured in a designed four-alpha-helix bundle, [Delta7-X10] 2. Quantitative agreement is obtained between theory and experiment for relative binding affinities and reduction potentials. Using this protocol, we develop relative scales of binding affinities for ferrous and ferric heme b as well as the reduction potential of the FeIII/FeII redox couple. It is found that Lys-Lys, His-Lys, Met-Met, and His-Met can make the reduction potential more positive than His-His. For Lys-Lys and His-Lys, the binding affinities for both oxidation states are enhanced with greater stabilization of ferrous heme b relative to ferric heme b. For Met-Met and His-Met, the binding affinities for both states decline with greater destabilization of ferric heme b as compared to ferrous heme b. Relative to His-His, Nature can make the reduction potential more negative with His-Cys-- and His-Tyr--. For these coordination motifs, the mechanisms behind this change are dependent on the dielectric constant of the environment. To test the accuracies of these calculations, we conducted thermodynamic characterizations on the following coordination motifs in cyt b562: His-Cys--, His-His, His-Lys, His-Met, and His-Tyr--. Since the set of axial ligands for cyt b562 is naturally His-Met, we can replace Met at position 7 with Cys-- (M7C), His (M7H), Lys (M7K), and Tyr-- (M7Y) to generate the coordination motifs above. Most of these variants are observed to bind ferrous and ferric heme b with the expected combination of axial ligands. In reduced holo M7C and M7Y, it is found that other coordination motifs besides His-Cys-- and His-Tyr-- are coordinating the ferrous iron of heme b. The calculated and experimental values for relative reduction potentials are in good agreement with each other. However, there are significant differences between calculated and experimental values of binding affinities for ferrous and ferric heme b. |
| Collection(s): | Doctoral Dissertations |
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