Weiss 9727980 SoxR of the bacterium Escherichia coli is a transcriptional activator for an oxidative stress regulon. It is one of only three known regulatory Fe-S proteins and the only one with a 2Fe-2S center. Ultimate goals of the study are (i) to find out how oxidation of the Fe-S clusters alters SoxR to become a transcriptional activator, and (ii) to elucidate the redox pathway that counters the autooxidation of its Fe-S clusters and keeps them in a reduced state in the uninduced cell. As an aid to the intended studies, SoxR derivatives will be produced containing deletions or protein fusions that may increase solubility and stability of this difficult protein. The protein will be dissected by deletion and point mutations in order to map specific domains and to see which ones might interact. These domains, whose functions are to be individually assayed, are ones involved in dimer formation, DNA binding, Fe-S cluster formation, reductase recognition, RNA polymerase binding, and open complex formation. Heterodimers of mutant and wild type proteins will be used to determine the possible participation of both polypeptide strands in some of these domains. In addition, the cellular reductase pathway for SoxR will be investigated by standard techniques of protein and cofactor fractionation. In addition, mutations for some possible genes in this pathway will be examined, and toward this end, insertion mutations will be produced for each of the two known flavodoxins. The SoxR protein of Escherichia coli regulates about 10 genes responding to oxidative stress. It can sense changes in the oxidation state of the cell, which are brought about by various oxidants such as the superoxide radical anion or nitric oxide. Superoxide is produced from oxygen by electron leakage during aerobic metabolism. It is also produced by some environmental toxicants like the herbicide paraquat and by macrophages as an antibacterial agent. The ultimate goal of this project is an understanding of cellular de fenses against highly reactive derivatives of oxygen, which is important, for example, in understanding how bacteria overcome plant defenses that involve oxidants.
