Determination of the pKa values of active-center cysteines, cysteines-32 and -35, in Escherichia coli thioredoxin by Raman spectroscopy.
We have determined the pKa values for thiol-thiolate equilibria of Escherichia coli thioredoxin and compared structural properties of reduced and oxidized forms of the protein in solution by Raman spectroscopy. Ionization and hydrogen-bonding states of the two cysteine sulfhydryls (Cys32 and Cys35) in reduced thioredoxin were determined by monitoring the complex Raman SH stretching band (Li & Thomas, 1991) as a function of pH in the range 4.0 < pH < 12.2. The Raman SH markers indicate the following: (i) Both sulfhydryls of the native protein are relatively robust hydrogen-bond donors, but one is a stronger hydrogen-bond donor than the other. (ii) The sulfhydryl which donates the weaker hydrogen bond, assigned tentatively to Cys32, is preferentially titrated to the thiolate ion (S-) as the solution pH is increased from 4 to 7. Water or a hydroxyl oxygen is the probable acceptor for the Cys32 S-H donor. (iii) The sulfhydryl which donates the stronger hydrogen bond, Cys35, resists substantial ionization until roughly 50% of the more acidic sulfhydryl has been titrated. A carbonyl oxygen is proposed as the likely acceptor of the Cys35 S-H donor. (iv) The Raman titration data indicate pK1 = 7.1 +/- 0.2 and pK2 = 7.9 +/- 0.2 for the two thiol-thiolate equilibria. The lower pKa, which is the more strongly perturbed, is assigned tentatively to Cys32.(ABSTRACT TRUNCATED AT 250 WORDS)
Li
H
H
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri, Kansas City 64110.
Hanson
C
C
Fuchs
J A
JA
Woodward
C
C
Thomas
G J
GJ
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012996:Solutions; D013059:Spectrum Analysis, Raman; D013879:Thioredoxins / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D005971:Glutamates; D012996:Solutions; D001224:Aspartic Acid; D018698:Glutamic Acid; D014443:Tyrosine; D013879:Thioredoxins; D014364:Tryptophan; D003545:Cysteine