Escherichia coli thioredoxin inhibition by cadmium: two mutually exclusive binding sites involving Cys32 and Asp26.

Françoise Rollin-Genetet, Catherine Berthomieu, Anne-Hélène Davin, Eric Quéméneur
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引用次数: 34

Abstract

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.

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镉对大肠杆菌硫氧还蛋白的抑制作用:涉及Cys32和Asp26的两个互斥结合位点。
观察到硫氧还蛋白对镉的抑制作用以及硫氧还蛋白对Cd(2+)的保护作用,使我们研究了硫氧还蛋白-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来探索活性位点内或附近的推定结合残基(Cys32、Cys35、Trp28、Trp31和Asp26)的相对贡献。在pH为8或7.5时,通过等温滴定量热法鉴定出两个结合位点,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,一个质子在Cd(2+)结合时被释放。在这些pH值下,用质谱法测定了每摩尔还原硫氧还蛋白中有1摩尔Cd(2+),表明两个结合位点被部分占据并且相互排斥。Cd(2+)在两个位点的结合完全抑制了Trx的巯基二硫转移酶活性。在氧化或烷基化Trx中检测到Cd(2+)相互作用的缺失以及Cd(2+)对硫氧还蛋白酶活性的抑制支持了Cys32在第一位点的作用。然而,Cd(2+)结合的硫氧还蛋白的荧光谱与氧化的硫氧还蛋白的荧光谱不同,表明Cd(2+)不与Cys32和Cys35配位。从FTIR光谱中,我们推断第二个位点可能涉及Asp26,这是一种埋藏的残留物,它以相当高且不寻常的pK(a)为羧酸盐(7.5/9.2)去质子化。两个残基Cys32和Asp26的pK(a)已被证明是相互依赖的[Chivers, t.p. (1997) biochemistry, 36, 14985-14991]。提出了一种机制,其中Cd(2+)结合在Cys32的溶剂可接近的硫代基上,诱导Asp26的pK(A)降低及其去质子化。相反,Asp26的羧酸基团与第二个结合位点的Cd(2+)相互作用诱导Cys32去质子化和硫氧还蛋白抑制,因此Cd(2+)不仅通过与Cys32结合,还通过与Asp26相互作用抑制硫氧还蛋白活性。
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