2004 Fiscal Year Final Research Report Summary
Elucidation of relationship between redox function and Fe-Met bond stability in thermostable cytochrome c
| Project/Area Number |
15550143
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Chemistry related to living body
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| Research Institution | University of Tsukuba |
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Principal Investigator |
YAMAMOTO yasuhiko University of Tsukuba, Graduate school of pure and applied sciences, Professor, 大学院・数理物質科学研究科, 教授 (00191453)
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| Project Period (FY) |
2003 – 2004
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| Keywords | Paramagnetic NMR / Heme complex / Metalloprotein / Electrochemistry / Coordination bond / Redox potential / Thermostability / Thermodynamics |
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| Research Abstract |
Understanding the molecular mechanisms responsible for regulation of the redox potentials (E°') of proteins is a problem of immense fundamental and practical importance. Monoheme Class I cytochromes c (cyts c), in which heme Fe is coordinated to His N and Met S atoms as axial ligands at the redox center, are some of the best characterized redox active proteins. Homologous Class I cyts c, i. e., thermophilic Hydrogenobacter thermophilus cytochrome c_<552> (HT) and mesophilic Pseudomonas aerugiaosa cytochrome c_<5512> (PA), exhibit a unique thermodynamic property, i. e., despite their structural similarity together with their 56 % sequence identity, the oxidized form of HT is significantly more stable than that of PA, as reflected by the large difference in denaturation temperature. Site-directed mutants of PA, for which amino acid substitutions were selected with reference to the corresponding residues in HT, exhibited thermostabilities between those of PA and HT.
PA and a series of its mutants exhibiting various thermostabilities have been studied by paramagnetic ^1H NMR and cyclic voltammetry in order to elucidate the molecular mechanisms responsible for control of the E°' value of the proteins. The study revealed that the E°' value of the protein is regulated by two molecular mechanisms operating independently of each other. One is based on the Fe-Met coordination bond strength in the protein, which is determined by the amino acid side-chain packing in the protein, and the other on the pKa value of the heme 17-propionic acid side-chain, which is affected by the electrostatic environment. The former mechanism alters the magnitude of the E°' value throughout the entire pH range and the latter regulates the pK values reflected by the pH profile of the E°' value. These findings provide novel insights into functional regulation of the protein, which could be utilized for tuning the E°' value of the protein by means of protein engineering.
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Research Products
(31 results)
