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Structures and Reactivities of Higher-valent Reaction Intermediates of Heme-containing Enzymes

含血红素酶的高价反应中间体的结构和反应活性

基本信息

批准号：
62480460
负责人：
MAKINO Ryu
金额：
$ 3.97万
依托单位：
Keio University, School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

Heme-containing enzymes yield nearly identical reaction intermediates such as Fe(III)-O2^- and Fe(IV)=O species. Nevertheless, the reactive oxygen in the intermediates was incorporated into the substrate in the reaction by cytochrome P450, while that is meraly reduced to water in the peroxidase reaction. To elucidate the reactivity difference, effects of the fifth axial and distal side amino acids of the heme-iron on the structure of the heme-bound ligand were analyzed by resonance Raman and infrared spectroscopic methods. The findings are summarized as follows; 1) From the comparison of the stretching frequencies of nitric oxide (NO) bound to cytochrome P450cam, chloroperoxidase (CPO) and horseradish peroxidase (HRP), it was suggested that the cystein thiolate as the fifth axial ligand in P450cam and CPO donated the electron density to the heme-bound ligand, thereby decreasing the N-O bond strength. 2) The presence of a dissociable amino acid residue was found to locate in the distal … More side of the heme in peroxidases (HRP and CPO), while that was absent in P450cam. Further, in a reaction intermediate, compound II, of HRP the distal amino acid residue was responsible for hydrogen-bond fromation with the oxygen of Fe(IV)=O, thereby regulating the reactivity of compound II. In all the enzymes examined, the addition of substrate affected the C-O stretching frequency of the ligand carbon monoxide, indicating that the bound-ligand interacted with the suvstrate. 3) The stretching vibration of the bound oxygen in the oxygenated form of CPO located at around 1130 cm^<-1> which essentially agreed with those of P450cam and myoglobin.These results suggest that the reactivity of the heme-bound oxygen, i.e. the function of the heme-enzymes is determined by the interaction with the amino acid residue in the distal side of the heme-iron, rather than the effect of the electron conating capacity of the fifth axial ligand. Interestingly, the O-O stretching frequency of oxygenated forms was found to be independent of an electronic character of the fifth axial amino acid residue. Less

含血红素的酶产生几乎相同的反应中间体，如Fe（III）-O2^-和Fe（IV）=O物质。然而，中间体中的活性氧在反应中被细胞色素P450掺入底物中，而在过氧化物酶反应中被还原成水。为了阐明反应性差异，血红素铁的第五轴向和远端侧氨基酸对血红素结合配体的结构的影响通过共振拉曼和红外光谱方法进行了分析。结果表明：（1）通过比较细胞色素P450 cam、氯过氧化物酶（CPO）和辣根过氧化物酶（HRP）与NO结合的伸缩频率，推测P450 cam和CPO中的第五轴配体巯基半胱氨酸将电子密度贡献给血红素结合配体，从而降低了N-O键的强度。2)发现存在一个可解离的氨基酸残基位于远端 ...更多信息过氧化物酶（HRP和CPO）在血红素的一侧有表达，而P450 cam则无表达。此外，在HRP的反应中间体化合物II中，远端氨基酸残基负责与Fe（IV）=O的氧形成氢键，从而调节化合物II的反应性。在所有酶中，底物的加入影响了配体一氧化碳的C-O伸缩频率，表明结合配体与底物相互作用。3)氧化态CPO中结合氧的伸缩振动位于1130 cm ~ 2附近，<-1>与P450 cam和肌红蛋白的伸缩振动基本一致，表明血红素结合氧的活性即血红素酶的功能是由与血红素铁远端氨基酸残基的相互作用决定的，而不是由第五轴配体的电子传递能力决定的。有趣的是，O-O伸缩频率的含氧形式被发现是独立的第五轴氨基酸残基的电子字符。少