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

含有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物种。然而，在细胞色素P450的反应中，中间产物中的活性氧被结合到底物中，而在过氧化物酶反应中，活性氧几乎被还原为水。为了解释反应活性的差异，用共振拉曼光谱和红外光谱方法分析了血红素-铁的第五轴向氨基酸和远侧氨基酸对血红素结合配体结构的影响。1)通过比较细胞色素P450cam、氯化过氧化物酶(CPO)和辣根过氧化物酶(HRP)与一氧化氮(NO)结合的伸展频率，推测P450cam和CPO中作为第五轴向配体的半胱氨酸硫酸酯为血红素结合配体提供了电子密度，从而降低了N-O键的强度。2)在…远端有一个游离氨基酸残基的存在在过氧化物酶(HRP和CPO)中有更多的血红素，而在P450cam中没有。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的反应活性。在所考察的所有酶中，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)CPO氧化态结合氧的伸缩振动位于1130 cm~(-1)~(-1)，与P450cam和肌红蛋白的伸缩振动基本一致。这些结果表明，血红素结合氧的反应性，即血红素酶的功能是由与血红素铁末端氨基酸残基的相互作用决定的，而不是第五轴向配体的电子连接能力的影响。有趣的是，含氧形式的O-O伸缩频率与第五轴向氨基酸残基的电子性质无关。较少

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