Selenomethionine-Substituted Thermus thermophilus Cytochrome ba3: Characterization of the CuA Site by Se and Cu K-EXAFS

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• 作者：Ninian J. Blackburn ; Martina Ralle ; Ester Gomez ; Michael G. Hill ; Andrzej Pastuszyn ; Donita Sanders ; and James A. Fee
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：June 1, 1999
• 年：1999
• 卷：38
• 期：22
• 页码：7075 - 7084
• 全文大小：125K
• 年卷期：v.38,no.22(June 1, 1999)
• ISSN：1520-4995

文摘

We have designed a gene that encodes a polypeptide corresponding to amino acids 44-168of the Thermus thermophilus cytochrome ba₃ subunit II [Keightley et al. (1995) J. Biol. Chem. 270,20345-20358]. The resulting ba₃-Cu_At10 protein separated into two fractions (A and B) during cationexchange chromatography which were demonstrated to differ only by N-terminal acetylation in fractionA. When the gene was expressed in an Escherichia coli strain that is auxotrophic for methionine andgrown in the presence of selenomethionine (Se(Met)), the single methionine of the Cu_At10 protein wasquantitatively replaced with Se(Met). Native (S(Met)) and Se(Met)-substituted proteins were characterizedby electrospray mass, optical absorption, and EPR spectroscopies and by electrochemical analysis; theywere found to have substantially identical properties. The Se(Met)-containing protein was furthercharacterized by Se and Cu K-EXAFS which revealed Cu-Se bond lengths of 2.55 Å in the mixed-valence form and 2.52 Å in the fully reduced form of Cu_A. Further analysis of the Se- and Cu-EXAFSspectra 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.