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Diethyl pyrocarbonate modification of the ligating histidines in the Cu A protein from Thermus thermophilus
Author(s) -
Hunsicker-Wang Laura
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.03777
Subject(s) - chemistry , histidine , thermus thermophilus , chemical modification , circular dichroism , electron paramagnetic resonance , stereochemistry , redox , porphyrin , cytochrome c oxidase , crystallography , biochemistry , amino acid , enzyme , organic chemistry , nuclear magnetic resonance , physics , escherichia coli , gene
The Cu A protein is subunit II of cytochrome oxidase, the terminal enzyme in the electron transport chain. The binuclear metal center of the Cu A protein is ligated by two bridging cysteines, two histidines and two weakly interacting ligands. Diethyl pyrocarbonate (DEPC) is a common chemical modifier that reacts with histidines, lysines, and the N‐terminus of proteins. DEPC was used to probe the reactivity of the ligating histidines of the Cu A protein to identify if one or both of them would be susceptible to modification. Visible circular dichroism and UV‐Visible spectroscopies were utilized to show the changes in the spectra upon modification and the data suggest that at least one of the ligating histidines becomes modified. Using fewer equivalents showed that as few as 5 equivalents results in modification of the ligating histidine. Electron paramagnetic resonance indicated that the cluster remains oxidized after modification. These results point to the role of the ligating histidines in modulating the redox properties of this cluster. Mononuclear proteins, including azurin and Sco, were also tested and these proteins do not display ligating histidines reactive toward DEPC. Thus, DEPC reactivity is not a property of all ligating histidines. Support or Funding Information Arnold and Mabel Beckman Foundation Beckman Scholars Program