Autocatalytic formation of green heme:: Evidence for H2O2-dependent formation of a covalent methionine-heme linkage in ascorbate peroxidase
Autocatalytic formation of green heme:: Evidence for H2O2-dependent formation of a covalent methionine-heme linkage in ascorbate peroxidase
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DOI：
10.1021/ja048242c
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发表时间：
2004-12-15
影响因子：
15
通讯作者：
Raven, EL
中科院分区：
文献类型：
--
作者：
Metcalfe, CL;Ott, M;Raven, EL
哺乳动物血红素过氧化物酶与它们的植物和真菌对应物的区别在于，血红素基团通过从谷氨酸和天冬氨酸残基到血红素1-和5-甲基的酯键共价结合到蛋白质，并且在髓过氧化物酶的情况下，通过从2-乙烯基的C-β到甲硫氨酸残基的另外的锍键共价结合到蛋白质。为了复制髓过氧化物酶中的硫连接并获得其形成机制的信息，我们设计了一个靠近重组豌豆胞质抗坏血酸过氧化物酶（rpAPX）中2-乙烯基的甲硫氨酸残基，通过Met（S160 M变体）替换Ser 160。S160 M变体作为脱辅基蛋白从大肠杆菌中分离。apo-S160 M与外源血红素的重组产生红色蛋白（S160 M（R）），其具有与rpAPX类似的UV-可见（λ（max）/nm = 407，511，633）和稳态动力学（k（cat）= 156 7s（-1），K-M = 102 +/- 15 μ M）性质。S160 MR与H_2O_2反应得到绿色蛋白（S160 M（G））。电子光谱、质谱和HPLC分析与S160 MG中蛋氨酸残基和血红素乙烯基之间形成共价键一致。单波长和光电二极管阵列停流动力学分析鉴定了作为反应中间体的瞬态化合物I物质。这些结果提供了第一个直接证据，即共价血红素连接形成作为涉及化合物I形成的H2 O2依赖性过程发生。提出了一种与数据一致的机制。
The mammalian heme peroxidases are distinguished from their plant and fungal counterparts by the fact that the heme group is covalently bound to the protein through ester links from glutamate and aspartate residues to the heme 1- and 5-methyl groups and, in the case of myeloperoxidase, through an additional sulfonium link from the C-beta of the 2-vinyl group to a methionine residue. To duplicate the sulfonium link in myeloperoxidase and to obtain information on its mechanism of formation, we have engineered a methionine residue close to the 2-vinyl group in recombinant pea cytosolic ascorbate peroxidase (rpAPX) by replacement of Ser160 by Met (S160M variant). The S160M variant is isolated from Escherichia coli as apo-protein. Reconstitution of apo-S160M with exogenous heme gives a red protein (S160M(R)) which has UV-visible (lambda(max)/nm = 407, 511, 633) and steady-state kinetic (k(cat) = 156 7 s(-1), K-M = 102 +/- 15 muM) properties that are analogous to those of rpAPX. The reaction of S160MR with H2O2 gives a green protein (S160M(G)). Electronic spectroscopy, mass spectrometry, and HPLC analyses are consistent with the formation of a covalent linkage between the methionine residue and the heme vinyl group in S160MG. Single-wavelength and photodiode array stopped-flow kinetic analyses identify a transient Compound I species as a reaction intermediate. The results provide the first direct evidence that covalent heme linkage formation occurs as an H2O2-dependent process that involves Compound I formation. A mechanism that is consistent with the data is presented.