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Title: Characterization and effect of metal ions on the formation of the Thermus thermophilus Sco mixed disulfide intermediate
Abstract The Sco protein fromThermus thermophilushas previously been shown to perform a disulfide bond reduction in the CuAprotein fromT. thermophilus, which is a soluble protein engineered from subunit II of cytochromeba3oxidase that lacks the transmembrane helix. The native cysteines onTtSco andTtCuAwere mutated to serine residues to probe the reactivities of the individual cysteines. Conjugation of TNB to the remaining cysteine inTtCuAand subsequent release upon incubation with the complementaryTtSco protein demonstrated the formation of the mixed disulfide intermediate. The cysteine ofTtSco that attacks the disulfide bond in the targetTtCuAprotein was determined to beTtSco 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 Cu2+or Cu+toTtSco prior to reaction withTtCuAwas found to preclude formation of the mixed disulfide intermediate. These results shed light on a mechanism of disulfide bond reduction by theTtSco protein and may point to a possible role of metal binding in regulating the activity. ImportanceThe function of Sco is at the center of many studies. The disulfide bond reduction in CuAby Sco is investigated herein and the effect of metal ions on the ability to reduce and form a mixed disulfide intermediate are also probed.  more » « less
Award ID(s):
1726441
PAR ID:
10076846
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Protein Science
Volume:
27
Issue:
11
ISSN:
0961-8368
Page Range / eLocation ID:
p. 1942-1954
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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