Exposure of Rhodospirillum rubrum to carbon monoxide (CO) stimulates the expression of a set of genes whose products oxidize CO to CO2 with concomitant reduction of H+ to H2. This response is regulated by the cooA (CO-oxidation Activator) gene product, CooA. A variety of genetic and physiological results indicate that CooA is an unusual member of the important cAMP Receptor Protein (CAP) family of transcriptional effectors. In particular, modeling the CooA sequence on the CAP structure suggests a metal cluster-binding motif in the region homologous to the cAMP binding site of CAP. Such clusters in proteins are often targets for CO-binding. The proposed research will elucidate the mechanism by which CooA mediates the response of R. rubrum to the presence of CO. The first specific aim will be to purify CooA, since this will be essential for any detailed understanding of its role in CO-sensing and response. Simultaneously we will develop an in vitro functional assay for CooA and a protocol for its overexpression. These tools will be used in concert with our expertise in anaerobic protein purification to purify mg quantities of functional CooA. The second specific aim will directly examine the CO-sensing domain of CooA. The purified protein will be analyzed for the presence of metals and metal clusters. The spectra of CooA both in the presence and absence of CO will be examined. The results will indicate the number and architecture of the metals at the CO-sensing motif, which will be further examined through the use of site-directed mutagenesis and subsequent biochemical analysis of a few selected mutant versions of CooA. The final aim will characterize the response of CooA to CO. This will involve examination of the conformation and oligomerization states of CooA, as well as parameters of DNA binding and transcriptional activation in the presence and absence of CO. Response-altered mutants will be found either through directed mutagenesis based on the CAP paradigm or through direct selection schemes that have already been developed. The result of this work will be an insight into the molecular basis for biological CO recognition as well as improved understanding of the CAP/FNR family. The elucidation of CO binding is important to our understanding of the effects of CO as a pollutant as well as its possible role as a biological signal molecule.