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首页> 外文期刊>Biochemistry >Spectroscopic Studies of Single and Double Variants of M Ferritin: Lack of Conversion of a Biferrous Substrate Site into a Cofactor Site for O_2 Activation
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Spectroscopic Studies of Single and Double Variants of M Ferritin: Lack of Conversion of a Biferrous Substrate Site into a Cofactor Site for O_2 Activation

机译：铁蛋白M的单变体和双变体的光谱研究：缺乏将亚铁底物位点转化为辅因子位点以进行O_2活化的功能

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Ferritin has a binuclear non-heme iron active site that functions to oxidize iron as a substrate for formation of an iron mineral core. Other enzymes of this class have tightly bound diiron cofactor sites that activate O_2 to react with substrate. Ferritin has an active site ligand set with 1-His/4- carboxylate/1-Gln rather than the 2-His/4-carboxylate set of the cofactor site. This ligand variation has been thought to make a major contribution to this biferrous substrate rather than cofactor site reactivity. However, the Q137E/D140H double variant of M ferritin, has a ligand set that is equivalent to most of the diiron cofactor sites, yet did not rapidly react with O_2 or generate the peroxy intermediate observed in the cofactor sites. Therefore, in this study, a combined spectroscopic methodology of circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD has been applied to evaluate the factors required for the rapid O_2 activation observed in cofactor sites. This methodology defines the coordination environment of each iron and the bridging ligation of the biferrous active sites in the double and corresponding single variants of frog M ferritin. Based on spectral changes, the D140H single variant has the new His ligand binding, and the Q137E variant has the new carboxylate forming a μ-1,3 bridge. The spectra for the Q137E/D140H double variant, which has the cofactor ligand set, however, reflects a site that is more coordinately saturated than the cofactor sites in other enzymes including ribonucleotide reductase, indicating the presence of additional water ligation. Correlation of this double variant and the cofactor sites to their O_2 reactivities indicates that electrostatic and steric changes in the active site and, in particular, the hydrophobic nature of a cofactor site associated with its second sphere protein environment, make important contributions to the activation of O_2 by the binuclear non-heme iron enzymes.
机译：铁蛋白具有双核非血红素铁活性位点，该活性位点起到氧化铁作为形成铁矿物质核的底物的作用。这类其他酶具有紧密结合的二铁辅因子位点，可激活O_2与底物反应。铁蛋白具有设置有1-His / 4-羧酸盐/ 1-Gln的活性位点配体，而不是辅因子位点的2-His / 4-羧酸盐组。人们认为这种配体变化是对该双亚铁底物的主要贡献，而不是辅因子位点的反应性。然而，铁蛋白M的Q137E / D140H双变体具有等同于大多数二铁辅因子位点的配体组，但并未与O_2快速反应或产生在辅因子位点中观察到的过氧中间体。因此，在这项研究中，圆二色性（CD）/磁性CD（MCD）/可变温度，可变场（VTVH）MCD的组合光谱方法已用于评估在辅因子位点观察到的O_2快速活化所需的因子。该方法定义了青蛙铁蛋白双和相应的单个变体中每个铁的配位环境和双铁活性位点的桥接连接。基于光谱变化，D140H单个变体具有新的His配体结合，而Q137E变体具有形成μ-1,3桥的新羧酸盐。具有辅因子配体集的Q137E / D140H双变异体的光谱反映的位点比其他酶（包括核糖核苷酸还原酶）中的辅因子位点更协调饱和，表明存在额外的水连接。此双变异体和辅助因子位点与其O_2反应性的相关性表明，活性位点的静电和空间变化，尤其是与其第二球蛋白环境相关的辅助因子位点的疏水性，对激活其活性做出了重要贡献。 O_2由双核非血红素铁酶组成。

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