Bis-methionine ligation to heme iron in mutants of cytochrome b(562) .1. Spectroscopic and electrochemical characterization of the electronic properties'
Bis-methionine ligation to heme iron in mutants of cytochrome b(562) .1. Spectroscopic and electrochemical characterization of the electronic properties'
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DOI：
10.1021/bi961127x
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发表时间：
1996-10-22
期刊：
影响因子：
2.9
通讯作者：
Hill, HAO
中科院分区：
文献类型：
--
作者：
Barker, PD;Nerou, EP;Hill, HAO
我们已经产生了细胞色素B（562）的突变体，其中血红素铁的组氨酸配体（His 102）被甲硫氨酸取代，所产生的蛋白质可以具有与血红素铁的双甲硫氨酸配位，但是这种排列的稳定性取决于氧化状态和溶液pH。和EPR光谱来研究在各种条件下血红素配位环境的性质。单一变体H102 M还原状态的光谱与双甲硫氨酸连接一致。在其氧化状态下，该蛋白质在所有研究条件下都是高自旋的，并且光谱性质与仅一个甲硫氨酸配体被配位一致。我们无法确定是什么提供了另一个轴向配体。双变体R98 C/H102 M（其中血红素通过C型硫醚键共价连接到蛋白质上）也是亚铁态的双甲硫氨酸配位，但在氧化态具有显著不同的性质。在20 ℃下pK（a）为7.1时，该蛋白质在低pH下从低自旋6配位血红素蛋白转化为高自旋物质，类似于在单一变体中观察到的高自旋物质。我们的光谱数据证明，低自旋物种是双甲硫氨酸协调。已使用直接电化学技术测量该双甲硫氨酸物质的还原电位，在pH 4.8时为+440 mV。这些蛋白质的电化学是复杂的耦合协调状态的变化。以下论文中提供的NMR结果证明亚铁态是双蛋氨酸配位的。
We have generated mutants of cytochrome b(562) in which the histidine ligand to the heme iron (His102) has been replaced by a methionine, The resulting proteins can have bis-methionine coordination to the heme iron, but the stability of this arrangement is dependent on oxidation stare and solution pH. We have used optical, MCD, and EPR spectroscopies to study the nature of the heme coordination environment under a variety of conditions. Optical spectra of the reduced state of the single variant, H102M, are consistent with bis-methionine ligation. In its oxidized state, this protein is high-spin under all conditions studied, and the spectroscopic properties are consistent with only one of the methionine ligands being coordinated. We cannot identify what, if anything, provides the other axial ligand. A double variant, R98C/H102M (in which the heme is covalently attached to the protein through a c-type thioether linkage), is also bis-methionine coordinated in the ferrous state, but has significantly different properties in the oxidized state. With a pK(a) of 7.1 at 20 degrees C, the protein converts from a low-spin, 6-coordinate heme protein at low pH, to a high-spin species, similar to the high-spin species observed for the single variant. Our spectroscopic data prove that the low-spin species is bis-methionine coordinated. The reduction potential of this bis-methionine species has been measured using direct electrochemical techniques and is +440 mV at pH 4.8. The electrochemistry of these proteins is complicated by coupled coordination-state changes. Proof that the ferrous state is bis-methionine coordinated is provided by NMR results presented in the following paper.