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Abstract The Sco protein fromThermus thermophilushas previously been shown to perform a disulfide bond reduction in the Cu_Aprotein fromT. thermophilus, which is a soluble protein engineered from subunit II of cytochromeba₃oxidase that lacks the transmembrane helix. The native cysteines onTtSco andTtCu_Awere mutated to serine residues to probe the reactivities of the individual cysteines. Conjugation of TNB to the remaining cysteine inTtCu_Aand 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 targetTtCu_Aprotein 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 withTtCu_Awas 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 Cu_Aby Sco is investigated herein and the effect of metal ions on the ability to reduce and form a mixed disulfide intermediate are also probed.