Loferer H, Wunderlich M, Hennecke H, Glockshuber R
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
J Biol Chem. 1995 Nov 3;270(44):26178-83. doi: 10.1074/jbc.270.44.26178.
The membrane-anchored thioredoxin-like protein (TlpA) from the Gram-negative soil bacterium Bradyrhizobium japonicum was initially discovered due to its essential role in the maturation of cytochrome aa3. A soluble form of TlpA lacking the N-terminal membrane anchor acts as a protein thiol:disulfide oxidoreductase. TlpA possesses an active-site disulfide bond common to all members of the thiol:disulfide oxidoreductase family. In addition, it contains two non-active-site cysteines that form a structural disulfide bond (Loferer, H., Bott, M., and Hennecke, H. (1993) EMBO J. 12, 3373-3383; Loferer, H., and Hennecke, H. (1994) Eur. J. Biochem. 223, 339-344). Here, we compare the far- and near-UV CD spectra of TlpA before and after reduction of both disulfides by dithiothreitol and show that the non-active-site disulfide bond is not required for the integrity of TlpA's native conformation. In contrast to dithiothreitol, reduced glutathione (GSH) selectively reduces the active-site disulfide and leaves the non-active-site disulfide bond intact, even at high molar excess over TlpA. The selective reduction of the active-site disulfide bond leads to a 10-fold increase of the intrinsic tryptophan fluorescence of TlpA at 355 nm, which may be interpreted as a quenching of tryptophan fluorescence by the active-site disulfide bond. Using the specific fluorescence of TlpA as a measure of its redox state, a value of 1.9 +/- 0.2 M was determined for the TlpA:glutathione equilibrium constant at pH 7.0, demonstrating that TlpA is a reductant, like cytoplasmic thioredoxins. The DsbA protein, which acts as the final oxidant of periplasmic secretory proteins in Escherichia coli, is not capable of oxidizing the active-site cysteines of TlpA. This suggests that TlpA's primary role in vivo is keeping the thiols of certain proteins reduced and that TlpA's active, reduced state may be maintained owing to its kinetically restricted oxidation by other periplasmic disulfide oxidoreductases such as DsbA.
来自革兰氏阴性土壤细菌日本慢生根瘤菌的膜锚定硫氧还蛋白样蛋白（TlpA）最初因其在细胞色素aa3成熟过程中的关键作用而被发现。一种缺乏N端膜锚定的可溶性TlpA形式可作为蛋白质硫醇：二硫键氧化还原酶。TlpA拥有硫醇：二硫键氧化还原酶家族所有成员共有的活性位点二硫键。此外，它还含有两个非活性位点的半胱氨酸，它们形成一个结构二硫键（Loferer，H.，Bott，M.，和Hennecke，H.（1993年）《欧洲分子生物学组织杂志》12卷，3373 - 3383页；Loferer，H.，和Hennecke，H.（1994年）《欧洲生物化学杂志》223卷，339 - 344页）。在此，我们比较了用二硫苏糖醇还原两个二硫键前后TlpA的远紫外和近紫外圆二色光谱，结果表明非活性位点二硫键对于TlpA天然构象的完整性并非必需。与二硫苏糖醇不同，即使在相对于TlpA有高摩尔过量的情况下，还原型谷胱甘肽（GSH）也能选择性地还原活性位点二硫键，而使非活性位点二硫键保持完整。活性位点二硫键的选择性还原导致TlpA在355 nm处的内在色氨酸荧光增加了10倍，这可能被解释为活性位点二硫键对色氨酸荧光的淬灭。以TlpA的特异性荧光作为其氧化还原状态的衡量指标，在pH 7.0时测定TlpA：谷胱甘肽平衡常数的值为1.9±0.2 M，这表明TlpA是一种还原剂，类似于细胞质硫氧还蛋白。在大肠杆菌中作为周质分泌蛋白最终氧化剂的DsbA蛋白，无法氧化TlpA的活性位点半胱氨酸。这表明TlpA在体内的主要作用是保持某些蛋白质的硫醇处于还原状态，并且TlpA的活性还原状态可能由于其被其他周质二硫键氧化还原酶（如DsbA）进行动力学限制的氧化而得以维持。
