Resonance Raman Evidence for the Presence of the FeIV=O Bond in Horseradish Peroxidase Compound II
Resonance Raman Evidence for the Presence of the FeIV=O Bond in Horseradish Peroxidase Compound II
复制标题
辣根过氧化物酶化合物 II 中存在 FeIV=O 键的共振拉曼证据
DOI：
--
复制
发表时间：
1984
期刊：
影响因子：
--
通讯作者：
T. Kitagawa
中科院分区：
文献类型：
--
作者：
S. Hashimoto;Y. Tatsuno;T. Kitagawa
辣根过氧化物酶（HRP）是一种含血红素的酶，其催化各种有机化合物与作为特异性氧化剂的过氧化氢氧化。催化作用通过H2 O2对铁酶的氧化和有机底物的连续还原进行。最初的氧化产生一种中间体，称为化合物I。其还原成天然铁态通过两个步骤经由称为化合物II的另一中间体进行。因此，化合物I和II具有比Fe+血红素分别高2和1个氧化当量的氧化态。对化合物Ⅱ的穆斯堡尔谱研究揭示了Fe'血红素的存在。关于血红素铁的第六配体存在争议;动力学研究3 ~表明Fe 1 °-OH结构，而ENDOR和NMR研究`>>表明Fein _ O结构。确定替代物是阐明该酶催化机制的关键。我们在这里描述了FeTP = 0结构的确凿证据，即FeW==0伸缩拉曼线的观测。HRP的同工酶C购自Toyobo（等级I-C r RZ = 3.2），并且不经进一步纯化而使用。将酶溶解在磷酸盐或甘氨酸缓冲液中，分别在中性或碱性pH下进行测量。先前描述了HRP与掺入~ 4Fe的血红素的重构。G>化合物II通过将等摩尔的H_2O_2和三价铁HRP混合而产生。通过在混合溶剂（二甲苯13.1，苯乙酮43.5，辛醇34.4wt%）中氧化2-乙基蒽醌与过量的18 O2（纯度99%），然后在氮气气氛下用脱气的H2O萃取来制备H218 O2的水溶液。2-乙基蒽醌在2 % Pd-A_（120）存在下原位加氢制得2-乙基蒽醌溶液。H21802的形成用其拉曼光谱证实，并且浓度为100 μ mol/L。
Horseradish peroxidase (HRP) is a heme-containing enzyme which catalyzes the oxidation of various organic compounds with hydrogen peroxide as a specific oxidant. The catalysis proceeds via the oxidation of the ferric enzyme by H202 and the successive reduction by the organic substrate. The initial oxidation gives rise to an intermediate called compound I. Its reduction to native ferric state is forwarded by two steps via the other intermediate called compound IL'~ Therefore, compounds I and II possess the higher oxidation states than the Fe" heme by two and one oxidation equivalents, respectively. Mossbauer studyon compound II revealed the presence of the Fe' heme. There have been arguments about the sixth ligand of the heme iron ; kinetic study3~ suggested the Fe1°-OH structure whereas the ENDOR' and NMR studies`>> suggested the Fein _ O structure. Determination of the alternatives is essential to elucidate the catalytic mechanism of this enzyme. We describe here a conclusive evidence for the FeTP = 0 structure, namely, the observationn of the FeW==0 stretching Raman line. Isozyme C of HRP was purchased from Toyobo (grade I-C r RZ = 3.2) and used without further purification. The enzyme was dissolved in phosphate or glycine buffer for the measurements at neutral or alkaline pH, respectively. Reconstitution of HRP with the ~4Fe-incorporated heme was described previously.G> Compound II was produced by mixing the equal moles of H2O2 and ferric HRP.. An aqueous solution of H21802 was prepared by oxidation of 2-ethyll anthraquinol in a mixed solvent (xylene 13.1, acetophenone 43.5, octanol 34.4 wt%) with an excess of 1802 (purity 99%), followed by extraction with degassed H2O under nitrogen atmosphere. The 2-ethyl anthraquinol solution was obtained in situ from hydrogenation of 2-ethyl anthraquinone in the presence of 2 % Pd-A120. The formation of H21802 was confirmed with its Raman spectrum, and the concen-