Disulfide dithiol redox titrations of proteins
Mason, Jeremy Todd
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Redox-active disulfide/dithiol couples in proteins play important regulatory roles within cells. Disulfide/dithiol redox reactions of regulatory proteins found in the purple photosynthetic bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus are used to regulate the expression of genes encoding photosystem components in response to the presence oxygen and light. Disulfide/dithiol redox reactions of regulatory proteins found in the yeast Saccharomyces cerevisiae regulate expression of genes encoding the production of peroxide-scavenging proteins in response to intracellular H2O2 tension. AppA and PpsR are two proteins present in R. sphaeroides that have been shown to regulate gene expression in response to oxygen through disulfide/dithiol redox chemistry. AppA, which contains FAD, also functions as a blue light receptor. It has been proposed that AppA reduces PpsR, causing PpsR to lose its abililty to bind DNA and repress transcription of photosynthesis genes. Redox titrations of the disulfide/dithiol couples in PpsR and AppA were carried out at pH 7.0 and the two proteins were shown to be isopotential, with both having Em values of -320 mV at pH 7.0. Em vs. pH profiles for PpsR and RegB, a protein involved in regulation of gene expression in R. capsulatus, were generated in an attempt to detect pKa values for groups involved in proton uptake/release that is coupled to the disulfide/dithiol redox chemistry. However, neither protein showed a pKa for redox-linked residues at physiological pH values. Yap1 is a key regulator of gene expression in S. cerevisiae in response to peroxides. Gpx3 and Trx2 are two additional components of this S. cerevisiae regulatory cascade. Em values for the two disulfide bonds in Yap1 have been determined (Em1 = -330 mV and Em2 = -155 mV), as has an Em value of -315 mV for Gpx3, a component thought to serve as the physiological oxidant for Yap1. Trx2 has an Em of -275 mV, which is capable of reducing the disulfide in Yap1 that corresponds to Em2, but not Em1.