Selenomethionine-substituted Thermus thermophilus cytochrome ba3: Characterization of the Cu(A) site by Se and Cu K-EXAFS

Ninian J. Blackburn, Martina Ralle, Ester Gomez, Michael G. Hill, Andrzej Pastuszyn, Donita Sanders, James A. Fee

Research output: Contribution to journalArticle

28 Scopus citations

Abstract

We have designed a gene that encodes a polypeptide corresponding to amino acids 44-168 of 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 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 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-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.

Original languageEnglish (US)
Pages (from-to)7075-7084
Number of pages10
JournalBiochemistry
Volume38
Issue number22
DOIs
StatePublished - Jun 1 1999

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Selenomethionine-substituted Thermus thermophilus cytochrome ba3: Characterization of the Cu(A) site by Se and Cu K-EXAFS'. Together they form a unique fingerprint.

  • Cite this