权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

STRUCTURAL STUDY OF THE DISTAL HISTIDINE IN PEROXIDASE BY RESONANCE RAMAN SPECTROSCOPY.

通过共振拉曼光谱研究过氧化物酶中远端组氨酸的结构。

基本信息

批准号：
15550021
负责人：
HASHIMOTO Shinji
金额：
$ 2.43万
依托单位：
SCIENCE UNIVERSITY OF TOKYO, YAMAGUCHI
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

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 … More 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

利用紫外共振拉曼光谱（UVRR）研究了辣根过氧化物酶（HRP）与一氧化碳（HRP-CO）复合物中组氨酸（His 42）的结构和氢键状态。HRP-CO-HRP UVRR差谱在pD7.0的D_2O溶液中显示两个分别位于1408和1388 cm ~ 2处的正峰<-1>，这两个峰分别归属于His 42的咪唑侧链的中弱和强氢键状态。His 42的两个峰强度随pD的增加而减弱，跃迁中点在pD8.8处，表明His 42在HRP-CO中的pJ为8.8。CO配体在1932和1902 cm-2处表现出两个C-0伸缩拉曼峰<-1>，后者在碱性pD处减小，并且可归属于强氢键状态。最有可能的是，His 42的咪唑钥侧链与CO形成强氢键，在一个共形物中在1388 cm-1处产生His 42峰<-1>，在1902 cm-1处产生CO峰。 ...更多信息 R. His 42的另一个氢键状态，给出1408 cm-1峰，归因于与溶剂水形成中等至弱氢键的另一种构象异构体。His 42可以作为血红素铁配体的氧原子的强质子供体的本发明发现支持His 42作为一般酸裂解过氧化氢（一种特异性氧化剂）的0-0键的作用，用共振拉曼光谱和红外光谱研究了辣根过氧化物酶的活性中心结构（不含Ca^<2+>的HRP）。氰化物结合形式的无Ca^2+ HRP的紫外共振拉曼差谱表明，末端组氨酸（His 42）在氰化物结合时被质子化。远端组氨酸的差谱的峰频率与天然HRP的峰频率相同，表明质子化和氢键状态没有受到结合的Ca 2+的去除的显着影响。HRP及其底物结合形式的红外光谱中CN伸缩带在Ca^2+耗尽后上移约1-2 cm^2<-1>。可能的是，配体拉伸模式的上移是由在血红素配体处的局部电场的变化引起的，所述局部电场的变化是由Ca 2+的去除引起的。少