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Conformation, stability, and active‐site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy
Author(s) -
Vohník Stanislav,
Tuma Roman,
Thomas George J.,
Hanson Chad,
Fuchs James A.,
Woodward Clare
Publication year - 1998
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.5560070120
Subject(s) - titration , raman spectroscopy , cysteine , thioredoxin , chemistry , escherichia coli , spectroscopy , crystallography , biochemistry , inorganic chemistry , physics , enzyme , optics , gene , quantum mechanics
The active‐site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low p K a values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. p K a values governing cysteine thiol‐thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent p K a values of 7.5 ± 0.2, close to values measured previously for wild‐type thioredoxin. Sulfhydryl Raman markers of D26A and wild‐type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side‐chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild‐type protein. Additionally, the thioredoxin main‐chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active‐site cysteines nor is responsible for their abnormally low p K a values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.