RELATIONSHIP BETWEEN ELECTROSTATICS AND REDOX FUNCTION IN HUMAN THIOREDOXIN - CHARACTERIZATION OF PH TITRATION SHIFTS USING 2-DIMENSIONAL HOMONUCLEAR AND HETERONUCLEAR NMR

The electrostatic behavior of potentially titrating groups in reduced human thioredoxin was investigated using two-dimensional (2D) H-1 and N-15 nuclear magnetic resonance (NMR) spectroscopy. A total of 241 chemical shift titration curves were measured over the pH range of 2.1-10.6 from homonuclear H-1-H-1 Hartmann-Hahn (HOHAHA) and heteronuclear H-1-N-15 Overbodenhausen correlation spectra. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the determination of pK(a)s for certain isolated ionizable groups, including the single histidine residue at position 43 (pK(a) = 5.5 +/- 0.1) and a number of aspartic and glutamic acid carboxylate groups. Many of the titration curves demonstrate complex behavior due to the effects of interacting titrating groups, the long range of electrostatic interactions through the protein interior, and, perhaps, pH-induced conformational changes on the chemical shifts. Unambiguous assignment of the pK(a)s for most of the 38 potentially ionizing groups of human thioredoxin could therefore not be made. In addition, there was no clear evidence that Asp-26 titrates in a manner corresponding to that observed in the Escherichia coli protein [Dyson, H. J., Tennant, L. L., & Holmgren, A. (I 99 1) Biochemistry 30, 4262-4268]. The pK(a)s of the active site cysteines were measured, however, with Cys-32 having an anomalously low value of 6.3 +/- 0.1 and that of Cys-35 between 7.5 and 8.6. These pK(a)s are in agreement with proposed mechanisms for redox catalysis of thioredoxin and previously measured pK(a)s within the active site of E. coli thioredoxin [Kallis, G. B., & Holmgren, A. (1980) J. Biol. Chem. 255, 10261-10265]. The stabilization of a thiolate anion at physiological pH can be explained by the interaction of the S(gamma) of Cys-32 with the amide of Cys-35 observed in the previously determined high-resolution solution structure of reduced human thioredoxin [Forman-Kay, J. D., Clore, G. M., Wingfield, P. T., & Gronenborn, A. M. (1991) Biochemistry 30, 2685-2698].