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

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-CuAt10 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 CuAt10 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 A in the mixed-valence form and 2.52 A 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.

Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary

“…The absorption bands for oxidized and reduced [SeMet 7 ]cyt b562 (oxidized, max ϭ 423, 533 nm, reduced, max ϭ 430, 533, 564 nm, Fig. 4C) were shifted compared with the analogous bands in wild-type cyt b562, consistent with observations of other metalloproteins that have sulfur to selenium substitutions in their ligand spheres (37)(38)(39). For instance, the replacement of the native methionine 80 with selenomethionine in cytochrome c by semisynthesis results in a red-shift in Soret absorbance in the oxidized form from 408 to 412 nm (37).…”

“…5). This Ϸ45 mV potential shift is in excellent agreement with that observed by Wallace and Clark-Lewis for their semisynthetic SeMet 80 cytochrome c mutant (37), whose potential is shifted by Ϫ48 mV compared with the methionineligated wild-type cytochrome c. This effect is much larger than that observed for the analogous Met 7 SeMet change in the purple CuA domain from cytochrome ba3; presumably, this difference is a result of the relatively strong bonding between heme iron and its axial ligands compared with the relatively weak copper methionine interaction in the CuA domain (38).…”

Anatomy of a Red Copper Center: Spectroscopic Identification and Reactivity of the Copper Centers of Bacillus subtilis Sco and Its Cys-to-Ala Variants

“…Cell cultures of the single variants (expressing S(Met)-containing proteins) were grown in 1 L LB-glucose medium containing 100 μg/mL of ampicillin at 37 °C to a final OD 600 between 0.6 and 0.8. Cell cultures of the double and triple SeM-containing variants were grown from the corresponding Met-minus strain B834(DE3) according to the earlier reported protocol 28. The cells were initially grown as a small culture (10 mL) in LB media with ampicillin overnight at 37 °C.…”

Crystal structures for short-chain pentraxin from zebrafish demonstrate a cyclic trimer with new recognition and effector faces

“…SeMet Dare-PTX was prepared using a reported method (Blackburn et al, 1999;Chen et al, 2010;Garboczi et al, 1996). The purification procedures for native and SeMet Dare-PTX were performed in accordance with the method described in our previous reports (Chen et al, 2010;Liu et al, 2012).…”