Rollin-Genetet Françoise, Berthomieu Catherine, Davin Anne-Hélène, Quéméneur Eric
CEA Valrhô, DSV-DIEP, Service de Biochimie postgénomique et Toxicologie Nucléaire, Bagnols-sur-Cèze, France.
Eur J Biochem. 2004 Apr;271(7):1299-309. doi: 10.1111/j.1432-1033.2004.04037.x.
Observations of thioredoxin inhibition by cadmium and of a positive role for thioredoxin in protection from Cd(2+) led us to investigate the thioredoxin-cadmium interaction properties. We used calorimetric and spectroscopic methods at different pH values to explore the relative contribution of putative binding residues (Cys32, Cys35, Trp28, Trp31 and Asp26) within or near the active site. At pH 8 or 7.5 two binding sites were identified by isothermal titration calorimetry with affinity constants of 10 x 10(6) m(-1) and 1 x 10(6) m(-1). For both sites, a proton was released upon Cd(2+) binding. One mole of Cd(2+) per mole of reduced thioredoxin was measured by mass spectrometry at these pH values, demonstrating that the two binding sites were partially occupied and mutually exclusive. Cd(2+) binding at either site totally inhibited the thiol-disulfide transferase activity of Trx. The absence of Cd(2+) interaction detected for oxidized or alkylated Trx and the inhibition of the enzymatic activity of thioredoxin by Cd(2+) supported the role of Cys32 at the first site. The fluorescence profile of Cd(2+)-bound thioredoxin differed, however, from that of oxidized thioredoxin, indicating that Cd(2+) was not coordinated with Cys32 and Cys35. From FTIR spectroscopy, we inferred that the second site might involve Asp26, a buried residue that deprotonates at a rather high and unusual pK(a) for a carboxylate (7.5/9.2). The pK(a) of the two residues Cys32 and Asp26 have been shown to be interdependent [Chivers, T. P. (1997) Biochemistry36, 14985-14991]. A mechanism is proposed in which Cd(2+) binding at the solvent-accessible thiolate group of Cys32 induces a decrease of the pK(a) of Asp26 and its deprotonation. Conversely, interaction between the carboxylate group of Asp26 and Cd(2+) at a second binding site induces Cys32 deprotonation and thioredoxin inhibition, so that Cd(2+) inhibits thioredoxin activity not only by binding at the Cys32 but also by interacting with Asp26.
镉对硫氧还蛋白的抑制作用以及硫氧还蛋白在抵御Cd(2+)方面的积极作用的观察结果，促使我们去研究硫氧还蛋白与镉的相互作用特性。我们在不同pH值下使用量热法和光谱法，以探究活性位点内或附近假定的结合残基（Cys32、Cys35、Trp28、Trp31和Asp26）的相对贡献。在pH 8或7.5时，通过等温滴定量热法鉴定出两个结合位点，其亲和常数分别为10×10(6) m(-1)和1×10(6) m(-1)。对于这两个位点，在Cd(2+)结合时都会释放一个质子。在这些pH值下，通过质谱法测得每摩尔还原型硫氧还蛋白结合1摩尔Cd(2+)，这表明两个结合位点是部分占据且相互排斥的。Cd(2+)在任一位点的结合都会完全抑制Trx的硫醇-二硫键转移酶活性。对于氧化型或烷基化的Trx未检测到Cd(2+)相互作用，以及Cd(2+)对硫氧还蛋白酶活性的抑制作用，都支持了Cys32在第一个位点的作用。然而，结合了Cd(2+)的硫氧还蛋白荧光图谱与氧化型硫氧还蛋白的不同，这表明Cd(2+)并非与Cys32和Cys35配位。从傅里叶变换红外光谱（FTIR）分析，我们推断第二个位点可能涉及Asp26，这是一个埋藏残基，其羧酸盐的pK(a)相当高且不寻常（7.5/9.2）。已证明Cys32和Asp26这两个残基的pK(a)是相互依存的[奇弗斯，T.P.（1997年）《生物化学》36，14985 - 14991]。提出了一种机制，其中Cd(2+)与Cys32的溶剂可及硫醇盐基团结合会导致Asp26的pK(a)降低并使其去质子化。相反，Asp26的羧酸盐基团与第二个结合位点的Cd(2+)之间的相互作用会诱导Cys32去质子化并导致硫氧还蛋白受到抑制，因此Cd(2+)不仅通过与Cys32结合，还通过与Asp26相互作用来抑制硫氧还蛋白的活性。
