| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2004: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
Site-directed mutations have been introduced into the tetraheme cytochrome subunit in the chimeric reaction center complex composed of that from the purple bacterium, Blastochioris viridis, and core subunits from other purple bacterium, Rubrivivax gelatinosus. About 50 mutants have been constructed and analyzed with methods of time-resolved spectroscopy. Results obtained are follows.
1. It was shown that the physiological electron donor, cytochrome c_2, donates an electron to the most distal heme from the core among the 4 hemes. When the cytochrome recognizes its reaction partner, electrostatic interaction between the surfaces of them is the most important driving force. The distribution of the charges important for the interaction was mapped on the structure of the tetraheme cytochrome.
2. The redox midpoint potentials of hemes in the tetraheme cytochrome were successfully changed up to 400mV by changing the net charges of the amino acid residues positioned near the hemes and away from the protein surface. Changing the amino acid working as an axial ligand to the heme iron, methionine, to cysteine caused about 450mV downshift in the midpoint potential.
3. The midpoint potentials of hemes are sequenced as -60, 310, 60 and 400mV from the outside to the core in the original chimeric reaction center. The highest potential heme in one of the mutants was lowered from 400mV to 0mV, resulted in a change of the redox profile of 4 hemes from low-high-low-high to low-high-low-low. In this mutant the rate of electron transfer to the chlorophyll dimer was about 100 times slower than that in the original chimera. This shows that the low-potential hemes intermediate the electron transfer in spite of their unfavorable energy level and supports a kinetic model that an electron is transferred through a thermodynamic "roller coaster" if a sufficient potential difference is kept between the end donor and the end acceptor.
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