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

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

Research output: Contribution to journalArticle

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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

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Selenomethionine
Thermus thermophilus
Methionine
Proteins
Genes
Acetylation
Structural Models
Bond length
Chromatography
Light absorption
Escherichia coli
Paramagnetic resonance
Cations
Spectrum Analysis
Spectroscopy
Amino Acids
Peptides
cytochrome ba3

ASJC Scopus subject areas

  • Biochemistry

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Selenomethionine-substituted Thermus thermophilus cytochrome ba3 : Characterization of the Cu(A) site by Se and Cu K-EXAFS. / Blackburn, Ninian; Ralle, Martina; Gomez, Ester; Hill, Michael G.; Pastuszyn, Andrzej; Sanders, Donita; Fee, James A.

In: Biochemistry, Vol. 38, No. 22, 01.06.1999, p. 7075-7084.

Research output: Contribution to journalArticle

Blackburn, Ninian ; Ralle, Martina ; Gomez, Ester ; Hill, Michael G. ; Pastuszyn, Andrzej ; Sanders, Donita ; Fee, James A. / Selenomethionine-substituted Thermus thermophilus cytochrome ba3 : Characterization of the Cu(A) site by Se and Cu K-EXAFS. In: Biochemistry. 1999 ; Vol. 38, No. 22. pp. 7075-7084.
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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 {\AA} in the mixed-valence form and 2.52 {\AA} 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.",
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