Selenomethionine-substituted Thermus thermophilus cytochrome ba3:: Characterization of the CuA site by Se and CuK-EXAFS
Selenomethionine-substituted Thermus thermophilus cytochrome ba3:: Characterization of the CuA site by Se and CuK-EXAFS
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DOI：
10.1021/bi982500z
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发表时间：
1999-06-01
期刊：
影响因子：
2.9
通讯作者：
Fee, JA
中科院分区：
文献类型：
--
作者：
Blackburn, NJ;Ralle, M;Fee, JA
我们设计了一个编码多肽的基因，对应于Thermus thermophilus细胞色素ba(3)亚基II的44-168个氨基酸。(1995)J.Biol.化学。20345-20358]。在阳离子交换层析过程中得到的ba(3)-Cu-AT10蛋白被分成两个组分(A和B)，这两个组分仅在组分A的N末端乙酰化不同。当该基因在对蛋氨酸营养缺乏的大肠杆菌中表达并在硒蛋氨酸(Se(Met))存在下生长时，铜-AT10蛋白的单一蛋氨酸被Se(Met)定量取代。用电喷雾质谱、光学吸收光谱、电子顺磁共振光谱和电化学分析对天然(S(蛋氨酸))和硒(蛋氨酸)取代的蛋白质进行了表征，发现它们具有基本相同的性质。对含Se(Met)的蛋白质进一步用Se和CuK-EXAFS进行了表征，得出混合价形式的铜-硒键长为2.55埃，完全还原形式的铜-硒键长为2.52埃。通过对Se-EXAFS和铜-EXAFS谱的进一步分析，得到了Se-S(硫酸盐)距离，从而得到了Se-Cu-Cu和Se-Cu-S(硫酸盐)角的信息。提出了扩展的EXAFS结构模型。
We have designed a gene that encodes a polypeptide corresponding to amino acids 44-168 of the Thermus thermophilus cytochrome ba(3) subunit II [Keightley et al. (1995) J. Biol. Chem. 270, 20345-20358]. The resulting ba(3)-Cu-At10 protein separated into two fractions (A and B) during cation exchange chromatography which were demonstrated to differ only by N-terminal acetylation in fraction A. When the gene was expressed in an Escherichia coli strain that is auxotrophic for methionine and grown in the presence of selenomethionine (Se(Met)), the single methionine of the Cu-At10 protein was quantitatively replaced with Se(Met). Native (S(Met)) and Se(Met)-substituted proteins were characterized by electrospray mass, optical absorption, and EPR spectroscopies and by electrochemical analysis; they were found to have substantially identical properties. The Se(Met)-containing protein was further characterized by Se and Cu K-EXAFS which revealed Cu-Se bond lengths of 2.55 Angstrom, in the mixed-valence form and 2.52 Angstrom in the fully reduced form of CUA Further analysis of the Se- and Cu-EXAFS spectra yielded the Se-S(thiolate) distances and thereby information on the Se-Cu-Cu and Se-Cu-S(thiolate) angles. An expanded EXAFS structural model is presented.