Spectroscopic characterization of site-specific [Fe(4)S(4)] cluster chemistry in ferredoxin:thioredoxin reductase: implications for the catalytic mechanism.
Spectroscopic characterization of site-specific [Fe(4)S(4)] cluster chemistry in ferredoxin:thioredoxin reductase: implications for the catalytic mechanism.
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DOI：
10.1021/ja051909q
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发表时间：
2005-07
影响因子：
15
通讯作者：
E. M. Walters;Ricardo García-Serres;Guy N. L. Jameson;Dominique A. Glauser;F. Bourquin;W. Manieri;P. Schürmann;Michael K. Johnson;B. Huynh
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文献类型：
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作者：
E. M. Walters;Ricardo García-Serres;Guy N. L. Jameson;Dominique A. Glauser;F. Bourquin;W. Manieri;P. Schürmann;Michael K. Johnson;B. Huynh
光调控的酶活性在产氧光合作用是介导的铁氧还蛋白：硫氧还蛋白还原酶（FTR），一类新的二硫化物还原酶的活性位点包括一个[Fe（4）S（4）]（2+）簇和相邻的二硫化物，催化还原硫氧还蛋白二硫化物在两个连续的单电子步骤使用[Fe（2）S（2）]（2+/+）铁氧还蛋白作为电子供体。在这项工作中，我们报告光谱（EPR，VTMCD，共振拉曼和穆斯堡尔）和氧化还原特性的活性位点的FTR在各种形式的酶，包括野生型FTR，点突变的变体在每个活性位点的半胱氨酸残基，和稳定的类似物的一个电子还原的FTR-Trx heterodisulfide中间体。结果揭示了新的特定位点的Fe（4）S（4）-簇化学氧化，单电子还原，和双电子还原形式的FTR。在静息酶中，Fe（4）S（4）簇和活性位点二硫键之间的弱相互作用促进了独特Fe位点的电荷积累，并引发活性位点接受来自铁氧还蛋白的电子以断开二硫键。在单电子还原类似物中，活性位点二硫化物的裂解伴随着形成活性位点二硫化物的半胱氨酸残基之一的配位，以产生在独特Fe位点具有两个半胱氨酸配体的[Fe（4）S（4）]（3+）簇。最有趣的结果是，其中二硫化物被还原为二硫醇的双电子还原FTR包含前所未有的富电子[Fe（4）S（4）]（2+）簇，其包含价离域和价定域的Fe（2+）Fe（3+）对。这些结果提供了分子水平的洞察FTR的催化机制，并提出了两个可行的机制。
Light regulation of enzyme activities in oxygenic photosynthesis is mediated by ferredoxin:thioredoxin reductase (FTR), a novel class of disulfide reductase with an active site comprising a [Fe(4)S(4)](2+) cluster and an adjacent disulfide, that catalyzes reduction of the thioredoxin disulfide in two sequential one-electron steps using a [Fe(2)S(2)](2+/+) ferredoxin as the electron donor. In this work, we report on spectroscopic (EPR, VTMCD, resonance Raman, and Mössbauer) and redox characterization of the active site of FTR in various forms of the enzyme, including wild-type FTR, point-mutation variants at each of the active-site cysteine residues, and stable analogues of the one-electron-reduced FTR-Trx heterodisulfide intermediate. The results reveal novel site-specific Fe(4)S(4)-cluster chemistry in oxidized, one-electron-reduced, and two-electron-reduced forms of FTR. In the resting enzyme, a weak interaction between the Fe(4)S(4) cluster and the active-site disulfide promotes charge buildup at a unique Fe site and primes the active site to accept an electron from ferredoxin to break the disulfide bond. In one-electron-reduced analogues, cleavage of the active-site disulfide is accompanied by coordination of one of the cysteine residues that form the active-site disulfide to yield a [Fe(4)S(4)](3+) cluster with two cysteinate ligands at a unique Fe site. The most intriguing result is that two-electron-reduced FTR in which the disulfide is reduced to a dithiol contains an unprecedented electron-rich [Fe(4)S(4)](2+) cluster comprising both valence-delocalized and valence-localized Fe(2+)Fe(3+) pairs. These results provide molecular level insights into the catalytic mechanism of FTR, and two viable mechanisms are proposed.