Thermodynamic Basis for Redox Regulation of the Yap1 Signal Transduction Pathway^†

The Yap1 oxidative stress signal transduction pathway found in Saccharomyces cerevisiae is redox-regulated. We have examined the thermodynamic basis of the disulfide/dithiol couples that are involved in the regulation of this pathway. The oxidized form of the Yap1 redox domain (Yap1-RD) fragment, derived from the Yap1 transcription factor, contains two disulfide bonds, one between Cys303 and Cys598 and one between Cys310 and Cys629. Oxidation−reduction titrations reveal the presence of two separate two-electron redox couples in Yap1-RD, with redox midpoint potentials (E_m) of −155 and −330 mV, respectively, at pH 7.0. We measured E_m values of −275 and −265 mV for the two cytoplasmic S. cerevisiae thioredoxins, Trx1 and Trx2, respectively, both at pH 7.0. Last, we measured an E_m value of −255 mV for the Cys36−Cys82 disulfide bond at pH 6.0 in the glutathione peroxidase-like enzyme, oxidant receptor protein (Orp1). We were unable to obtain satisfactory redox titration data for Orp1 at pH 7.0, but if the redox-active disulfide of Orp1 exhibits the −59 mV per pH unit dependence for E_m typical of protein disulfides in this pH region, an E_m value of −315 mV can be estimated for Orp1 at pH 7.0 by extrapolation. Together, these data suggest that, at physiological ratios of Trx_ox/Trx_red, the reduction of both the E_m = −315 mV disulfide of Orp1 and the E_m = −330 mV disulfide of Yap1 by either Trx1 or Trx2 would be thermodynamically possible.