2005 Fiscal Year Final Research Report Summary
STRUCTURAL STUDY OF THE DISTAL HISTIDINE IN PEROXIDASE BY RESONANCE RAMAN SPECTROSCOPY.
| Project/Area Number |
15550021
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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 |
Physical chemistry
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| Research Institution | SCIENCE UNIVERSITY OF TOKYO, YAMAGUCHI |
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Principal Investigator |
HASHIMOTO Shinji SCIENCE UNIVERSITY OF TOKYO, YAMAGUCHI, FACULTY OF SCIENCE AND ENGINEERING, PROFESSER, 基礎工学部, 教授 (50192696)
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| Project Period (FY) |
2003 – 2005
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| Keywords | heme protein / ultraviolet resonance Raman / peroxidase / enzymic activity / heydrogen peroxide / histidine / infrared spectroscopy |
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| Research Abstract |
Ultraviolet resonance Raman (UVRR) spectroscopy has been used to characterize the structure and hydrogen-bonding state of the distal histidine (His42) in horseradish peroxidase (HRP) complexed with carbon monoxide (HRP-CO). The HRP-CO-HRP UVRR difference spectrum in D_2O solution at pD 7.0 shows two positive peaks at 1408 and 1388 cm^<-1>, which are ascribable to medium-to-weak and strong hydrogen bonding states, respectively, of the protonated imidazolium side chain of His42 in HRP-CO. Both His42 peaks decrease in intensity with increase of pD with a midpoint of transition at pD 8.8, indicating that the pJ of His42 in HRP-CO is 8.8. The CO ligand exhibits two C-0 stretching Raman peaks at 1932 and 1902 cm^<-1>, the latter of which diminishes at alkaline pD and is assignable to a strongly hydrogen bonded state. It is most probable that the imidazolium side chain of His42 forms a strong hydrogen bond with CO, giving a His42 peak at 1388 cm^<-1> and a CO peak at 1902 cm', in one conforme
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r. The other hydrogen bonding state of His42, giving the 1408 cm-1 peak, is ascribed to another conformer forming a medium-to-weak hydrogen bond with solvent water. The present finding that His42 can act as a strong proton donor to the oxygen atom of the heme iron ligand gives support to the role of His42 as a general acid to cleave the 0-0 bond of hydrogen peroxide, a specific oxidizing agent, in the catalytic cycle of HRP.
The resonance Raman and infrared spectra were investigated to elucidate the active site structures of the calcium-depleted form of Horseradish peroxidase (Ca^<2+>-free HRP). The UV resonance Raman difference spectra of the cyanide-bound forms of Ca^<2+>-free HRP showed that the distal histidine (His42) was protonated upon the binding of cyanide. The peak frequencies of the difference spectrum of the distal histidine were same as those of native HRP, indicating that the protonation and hydrogen bonding states were not significantly affected by the removal of the bound Ca2. The CN stretch bands in the IR spectra of HRP and its substrate bound forms upshifted about 1-2 cm^<-1> upon the Ca^<2+> depletion. Possibly, the upshifts of the ligand stretch mode are caused by a change in the local electric field at the heme ligand caused by the removal of Ca2+. Less
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