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)