How Periplasmic Thioredoxin TlpA Reduces Bacterial Copper Chaperone ScoI and Cytochrome Oxidase Subunit II (CoxB) Prior to Metallation
How Periplasmic Thioredoxin TlpA Reduces Bacterial Copper Chaperone ScoI and Cytochrome Oxidase Subunit II (CoxB) Prior to Metallation
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DOI：
10.1074/jbc.m114.607127
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发表时间：
2014-11-21
影响因子：
4.8
通讯作者：
Glockshuber, Rudi
中科院分区：
文献类型：
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作者：
Abicht, Helge K.;Schaerer, Martin A.;Glockshuber, Rudi
细菌细胞色素c氧化酶亚基II（CoxB）上铜A位点（Cu-A）的两个关键半胱氨酸残基位于细胞质膜的周质侧。由于与还原性细胞质相比，周质是氧化环境，因此预测这些半胱氨酸之间形成的二硫键必须在铜插入之前通过还原消除。我们在这里表明，周质硫氧还蛋白（TlpA）作为一个特定的还原剂不仅为铜2+转移伴侣ScoI，但也为CoxB。动力学捕获的TlpA-ScoI和TlpA-CoxB混合二硫化物中间体的高分辨率晶体结构最好地证明了TlpA的双重作用。他们发现了两种蛋白质底物在TlpA上的不同接触位点。TlpA对CoxB的还原平衡显示了一个化学上有利的反应，与TlpA（E-0 '=-256 mV）相比，CoxB的负氧化还原电位（E-0'=-231 mV）较小。TlpA通过二硫键交换还原CoxB的速度非常快，速率常数为8.4 x 10（4）M-1 s（-1），与之前发现的ScoI还原相似。因此，TlpA是ScoI和CoxB的生理相关还原酶。虽然在体内，高浓度的环境Cu 2+可以绕过ScoI组装Cu-A-CoxB复合物的要求，但TlpA在这个过程中是必不可少的，因为只有还原的CoxB才能结合铜离子。
Two critical cysteine residues in the copper-A site (Cu-A) on subunit II (CoxB) of bacterial cytochrome c oxidase lie on the periplasmic side of the cytoplasmic membrane. As the periplasm is an oxidizing environment as compared with the reducing cytoplasm, the prediction was that a disulfide bond formed between these cysteines must be eliminated by reduction prior to copper insertion. We show here that a periplasmic thioredoxin (TlpA) acts as a specific reductant not only for the Cu2+ transfer chaperone ScoI but also for CoxB. The dual role of TlpA was documented best with high-resolution crystal structures of the kinetically trapped TlpA-ScoI and TlpA-CoxB mixed disulfide intermediates. They uncovered surprisingly disparate contact sites on TlpA for each of the two protein substrates. The equilibrium of CoxB reduction by TlpA revealed a thermodynamically favorable reaction, with a less negative redox potential of CoxB (E-0' = -231 mV) as compared with that of TlpA (E-0' = -256 mV). The reduction of CoxB by TlpA via disulfide exchange proved to be very fast, with a rate constant of 8.4 x 10(4) M-1 s(-1) that is similar to that found previously for ScoI reduction. Hence, TlpA is a physiologically relevant reductase for both ScoI and CoxB. Although the requirement of ScoI for assembly of the Cu-A-CoxB complex may be bypassed in vivo by high environmental Cu2+ concentrations, TlpA is essential in this process because only reduced CoxB can bind copper ions.