Setterdahl Aaron T, Chivers Peter T, Hirasawa Masakazu, Lemaire Stéphane D, Keryer Eliane, Miginiac-Maslow Myroslawa, Kim Sung-Kun, Mason Jeremy, Jacquot Jean-Pierre, Longbine Christopher C, de Lamotte-Guery Frédéric, Knaff David B
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79401-1061, USA.
Biochemistry. 2003 Dec 23;42(50):14877-84. doi: 10.1021/bi0302088.
Oxidation-reduction midpoint potential (E(m)) versus pH profiles were measured for wild-type thioredoxins from Escherichia coli and from the green alga Chlamydomonas reinhardtii and for a number of site-directed mutants of these two thioredoxins. These profiles all exhibit slopes of approximately -59 mV per pH unit, characteristic of the uptake of two protons per reduction of an active-site thioredoxin disulfide, at acidic, neutral, and moderately alkaline pH values. At higher pH values, these profiles exhibit slopes of either -29.5 mV per pH unit, characteristic of the uptake of one proton per disulfide reduced, or are pH-independent, indicating that neither proton uptake nor proton release is associated with reduction of the active-site disulfide. Reduction of the two wild-type thioredoxins is accompanied by the uptake of two protons even at pH values where the more acidic cysteine thiol group of the reduced proteins would be expected to be completely unprotonated. The effect of site-directed mutagenesis of two highly conserved aspartate residues that play important structural and/or catalytic roles in both thioredoxins, and which could in principle play a role in proton transfer, on the pK(a) values of redox-linked acid dissociations (deduced from changes in slope of the E(m) versus pH profiles) has also been determined for both E. coli thioredoxin and C. reinhardtii thioredoxin h.
测定了来自大肠杆菌和绿藻莱茵衣藻的野生型硫氧还蛋白以及这两种硫氧还蛋白的多个定点突变体的氧化还原中点电位（E(m)）与pH的关系曲线。在酸性、中性和中等碱性pH值下，这些曲线在每个pH单位上均呈现出约-59 mV的斜率，这是活性位点硫氧还蛋白二硫键每次还原吸收两个质子的特征。在较高pH值下，这些曲线呈现出每个pH单位-29.5 mV的斜率，这是每个二硫键还原吸收一个质子的特征，或者与pH无关，表明活性位点二硫键的还原既不伴随质子吸收也不伴随质子释放。即使在还原蛋白中酸性更强的半胱氨酸硫醇基团预计完全未质子化的pH值下，两种野生型硫氧还蛋白的还原也伴随着两个质子的吸收。还测定了对大肠杆菌硫氧还蛋白和莱茵衣藻硫氧还蛋白h中两个高度保守的天冬氨酸残基进行定点诱变对氧化还原相关酸解离的pK(a)值（从E(m)与pH曲线的斜率变化推导得出）的影响，这两个天冬氨酸残基在两种硫氧还蛋白中都起着重要的结构和/或催化作用，并且原则上可能在质子转移中发挥作用。
