Characterization and effect of metal ions on the formation of the Thermus thermophilus Sco mixed disulfide intermediate

Abstract

The Sco protein from Thermus thermophilus has previously been shown to perform a disulfide bond reduction in the Cu protein from T. thermophilus, which is a soluble protein engineered from subunit II of cytochrome ba oxidase that lacks the transmembrane helix. The native cysteines on TtSco and TtCu were mutated to serine residues to probe the reactivities of the individual cysteines. Conjugation of TNB to the remaining cysteine in TtCu and subsequent release upon incubation with the complementary TtSco protein demonstrated the formation of the mixed disulfide intermediate. The cysteine of TtSco that attacks the disulfide bond in the target TtCu protein was determined to be TtSco Cysteine 49. This cysteine is likely more reactive than Cysteine 53 due to a higher degree of solvent exposure. Removal of the metal binding histidine, His 139, does not change MDI formation. However, altering the arginine adjacent to the reactive cysteine in Sco (Arginine 48) does alter the formation of the MDI. Binding of Cu or Cu to TtSco prior to reaction with TtCu was found to preclude formation of the mixed disulfide intermediate. These results shed light on a mechanism of disulfide bond reduction by the TtSco protein and may point to a possible role of metal binding in regulating the activity. IMPORTANCE: The function of Sco is at the center of many studies. The disulfide bond reduction in Cu by Sco is investigated herein and the effect of metal ions on the ability to reduce and form a mixed disulfide intermediate are also probed.

Arabidopsis thaliana Hcc1 is a Sco‐like metallochaperone for CuA assembly in Cytochrome c Oxidase

“…The thiol-reductase activity of the Sco proteins was originally suggested upon the report of the crystal structure of B. subtilis Sco disclosing the existence of a mixed redox form (Sco 2SH and Sco SS ) [41]. This redox activity was later demonstrated biochemically for the Sco protein from T. thermophilus [35,57], which is able to reduce the oxidized cysteine residues of CoxII from the same organism. A similar reactivity was reported for Cu +1 -bound human Sco2, while the apo variant of human Sco2 was devoid of this activity [36].…”

DEPC modification of the CuA protein from Thermus thermophilus

“…The TtCu A protein and H40A/H117A were expressed and purified as was described previously for TtCu A [9], with a few minor changes. First, the expression plasmid contained an ampicillin resistance gene instead of a kanamycin gene, and is the parent plasmid used previously to study TtSco function [21]. Secondly, the sample regularly needed further purification after the CM column.…”

“…pET30 plasmids containing kanamycin resistance and His-tagged TtSco or H6A/H100A were grown and purified as was described for other TtSco mutants previously [21], except that MMTS was not added to the lysis mixture to cap the thiols. The proteins were quantified using the Edelhoch method [22,23].…”

“…9a). Sco has been proposed to assist in the formation of the Cu A site in CytcO by reducing the disulfide bond in apo TtCu A before the insertion of copper, though Sco's exact function is still debated and may vary across species [21,35,39,[45][46][47][48][49]. It binds one copper ion, which is ligated by two cysteines and one histidine (Fig.…”