| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2003: ¥3,000,000 (Direct Cost: ¥3,000,000)
|
|---|
| 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.
|
