Department of Chemistry, Stanford University , Stanford, California 94305, United States.
Biochemistry. 2014 Jan 28;53(3):473-82. doi: 10.1021/bi4013726. Epub 2014 Jan 15.
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 O2 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 O2 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 O2 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 O2 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 O2 by the binuclear non-heme iron enzymes.
铁蛋白具有双核非血红素铁活性位点，可作为形成铁矿物核心的底物氧化铁。该类别的其他酶具有紧密结合的二铁辅因子位点，可激活 O2 与底物反应。铁蛋白的活性位点配体具有 1-His/4-羧酸盐/1-Gln 而非辅因子位点的 2-His/4-羧酸盐。这种配体变化被认为是对这种双核底物而不是辅因子位点反应性的主要贡献。然而，M 铁蛋白的 Q137E/D140H 双变体具有与大多数二铁辅因子位点等效的配体集，但不能与 O2 快速反应或生成在辅因子位点中观察到的过氧中间体。因此，在这项研究中，采用圆二色性（CD）/磁圆二色性（MCD）/变温、变场（VTVH）MCD 的组合光谱方法来评估在辅因子位点中观察到的快速 O2 激活所需的因素。该方法定义了每个铁的配位环境以及蛙类 M 铁蛋白的双变体和相应的单变体中二价铁活性位点的桥接配位。基于光谱变化，D140H 单变体具有新的 His 配体结合，而 Q137E 变体形成新的羧酸盐形成 μ-1,3 桥。然而，具有辅因子配体集的 Q137E/D140H 双变体的光谱反映出比包括核苷酸还原酶在内的其他酶的辅因子位点更配位饱和的位点，表明存在额外的水配位。将这种双变体和辅因子位点与其 O2 反应性相关联表明，活性位点的静电和空间变化，特别是与辅因子位点相关的第二球蛋白质环境的疏水性，对双核非血红素铁酶的 O2 激活有重要贡献。
