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Characterization and effect of metal ions on the formation of the Thermus thermophilus Sco mixed disulfide intermediate
Author(s) -
Lopez Liezelle C.,
Mukhitov Nikita,
Handley Lindsey D.,
Hamme Cristina S.,
Hofman Cristina R.,
Euers Lindsay,
McKinney Jennifer R.,
Piers Amani D.,
Wadler Ellen,
HunsickerWang Laura M.
Publication year - 2018
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.3502
Subject(s) - thermus thermophilus , cysteine , chemistry , histidine , serine , stereochemistry , amino acid , biochemistry , enzyme , escherichia coli , gene
The Sco protein from Thermus thermophilus has previously been shown to perform a disulfide bond reduction in the Cu A protein from T. thermophilus , which is a soluble protein engineered from subunit II of cytochrome ba 3 oxidase that lacks the transmembrane helix. The native cysteines on Tt Sco and Tt Cu A were mutated to serine residues to probe the reactivities of the individual cysteines. Conjugation of TNB to the remaining cysteine in Tt Cu A and subsequent release upon incubation with the complementary Tt Sco protein demonstrated the formation of the mixed disulfide intermediate. The cysteine of Tt Sco that attacks the disulfide bond in the target Tt Cu A protein was determined to be Tt Sco 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 2+ or Cu + to Tt Sco prior to reaction with Tt Cu A was found to preclude formation of the mixed disulfide intermediate. These results shed light on a mechanism of disulfide bond reduction by the Tt Sco 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 A 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.