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

利用紫外共振拉曼光谱（UVRR）表征了辣根过氧化物酶（HRP）与一氧化碳（HRP- co）络合的远端组氨酸（His42）的结构和氢键状态。在pD 7.0的D_2O溶液中，HRP-CO- hrp UVRR差谱在1408和1388 cm^<-1>处显示出两个正峰，分别归因于HRP-CO中His42的质子化咪唑侧链的中弱和强氢键状态。随着pD的增加，两个His42峰的强度都降低，中间过渡点在pD 8.8处，表明hsp - co中His42的pJ为8.8。CO配体在1932和1902 cm^<-1>处表现出两个C-0拉伸拉曼峰，后者在碱性pD下减弱，属于强氢键态。最有可能的是，His42的咪唑侧链与CO形成了一个强氢键，在1388 cm^<-1 b>处有一个His42峰，在1902 cm'处有一个CO峰。His42的另一个氢键状态，在1408 cm-1处有一个峰，是由于另一个构象与溶剂水形成了一个中弱氢键。目前发现，His42可以作为血红素铁配体氧原子的强质子供体，这支持了His42在HRP的催化循环中作为一般酸裂解过氧化氢（一种特定的氧化剂）的0-0键的作用。研究了共振拉曼光谱和红外光谱，以阐明缺钙形式的辣根过氧化物酶（Ca^<2+> free HRP）的活性位点结构。无Ca^<2+>的HRP的氰化结合形式的紫外共振拉曼差分光谱表明，远端组氨酸（His42）在氰化结合后被质子化。远端组氨酸的差谱峰频率与天然HRP的差谱峰频率相同，表明去除结合Ca2对质子化和氢键状态没有显著影响。当Ca^<2+>耗尽时，HRP及其底物结合形式的红外光谱中的CN拉伸带向上移动约1-2 cm^<-1>。可能，配体拉伸模式的上升是由Ca2+的去除引起血红素配体局部电场的变化引起的。少

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