Spectroscopic studies of metal binding and metal selectivity in Bacillus subtilis BSco, a homologue of the yeast mitochondrial protein scolp
Spectroscopic studies of metal binding and metal selectivity in Bacillus subtilis BSco, a homologue of the yeast mitochondrial protein scolp
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DOI：
10.1021/ja0529539
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发表时间：
2005-11-30
影响因子：
15
通讯作者：
Blackburn, NJ
中科院分区：
文献类型：
--
作者：
Andruzzi, L;Nakano, M;Blackburn, NJ
Sco 1是一种线粒体膜蛋白，参与细胞色素c氧化酶CUA位点的组装。枯草芽孢杆菌基因组含有酵母Sco（1）的同源物YpmQ（以下称为BSco），其缺失导致缺乏caa（3）（含CUA）氧化酶活性但表达正常水平的aa（3）（醌醇）氧化酶活性的表型。在这里，我们报告的表征BSco的金属结合位点在其Cu（I），Cu（II），Zn（II），和Ni（II）的结合形式。发现Apo BSco以1：1的蛋白质/金属比率结合Cu（II）、Zn（II）和Ni（II）。Cu（I）蛋白可以通过Cu（II）衍生物的连二亚硫酸盐还原或通过用Cu（I）重构载脂蛋白来制备。X射线吸收光谱（XAS）表明Cu（I）在2.22 +/- 0.01埃处与两个半胱氨酸配位，在1.95 +/- 0.03埃处与弱结合的低Z散射体配位。Cu（II）衍生物是红橙色的，并且在350 nm附近表现出强的2型硫醇盐到Cu（II）的转变。多频电子顺磁共振（EPR），电子-核双共振（ENDOR），和电子自旋回波包络调制（ESEEM）的Cu（II）衍生物的研究提供了一个强耦合的组氨酸残基，至少一个强耦合的半胱氨酸，耦合到一个可交换的质子的证据。XAS光谱表明在2.21埃处有两个半胱氨酸配体，在1.95埃处有两个O/N供体配体，其中至少一个来自配位的组氨酸。Zn（II）和Ni（II）的衍生物均为4配位的MS 2N（His）X配位。这些结果提供的证据表明，铜分子伴侣可以从事氧化还原化学在金属中心，并可能建议有趣的氧化还原为基础的机制，金属化的混合价CUA中心的细胞色素c氧化酶。
Sco1 is a mitochondrial membrane protein involved in the assembly of the CUA site of cytochrome c oxidase. The Bacillus subtilis genome contains a homologue of yeast Sco(1), YpmQ (hereafter termed BSco), deletion of which leads to a phenotype lacking in caa(3) (CUA-containing) oxidase activity but expressing normal levels of aa(3) (quinol) oxidase activity. Here, we report the characterization of the metal binding site of BSco in its Cu(I)-, Cu(II)-, Zn(II)-, and Ni(II)-bound forms. Apo BSco was found to bind Cu(II), Zn(II), and Ni(II) at a 1:1 protein/metal ratio. The Cu(l) protein could be prepared by either dithionite reduction of the Cu(II) derivative or by reconstitution of the apo protein with Cu(I). X-ray absorption (XAS) spectroscopy showed that Cu(I) was coordinated by two cysteines at 2.22 +/- 0.01 angstrom and by a weakly bound low-Zscatterer at 1.95 +/- 0.03 angstrom. The Cu(II) derivative was reddish-orange and exhibited a strong type-2 thiolate to Cu(II) transition around 350 nm. Multifrequency electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and electron spin-echo envelope modulation (ESEEM) studies on the Cu(II) derivative provided evidence of one strongly coupled histidine residue, at least one strongly coupled cysteine, and coupling to an exchangeable proton. XAS spectroscopy indicated two cysteine ligands at 2.21 angstrom and two O/N donor ligands at 1.95 A, at least one of which is derived from a coordinated histidine. The Zn(II) and Ni(II) derivatives were 4-coordinate with MS2N(His)X coordination. These results provide evidence that a copper chaperone can engage in redox chemistry at the metal center and may suggest interesting redox-based mechanisms for metalation of the mixed-valence CUA center of cytochrome c oxidase.