| CooA from Rhodospirillum rubrum is the first CO-sensing protein discovered and provides an important model for other gas-sensing heme proteins. CooA binds to the regulatory element of the coo operon in the presence of CO, triggering the production of proteins that allow R. rubrum to utilize CO as the sole energy source. CooA was cloned and overexpressed in E. coli and purified by Dan Shelver in Prof. Gary Robert's laboratory at UW-Madison. In order to understand the mechanism of CO-activation, we characterized the heme-binding site of wild-type CooA using EPR and electronic absorption spectroscopy, pH dependence experiments and small ligand binding studies with cyanide, azide, imidazole, N-methylimidazole, thiol-modifying agents and NO. The data revealed that CooA contains a novel low-spin, cysteine-ligated, ferric heme. Only NO and thiol-modifying agents affected the electronic spectra of CooA, supporting the proposed coordination environment of the heme. EPR studies revealed that Cys75 is bound to the heme iron in Fe(III)CooA and that His77 interacts with Cys75. The current proposal is that His77 replaces Cys75 upon reduction to Fe(II)CooA and that His77-imidazolate is then displaced by CO. The displacement of a protein ligand by CO may lead to a conformational change in CooA.;The nitric oxide (NO) activation of sGC (soluble guanylyl cyclase) plays a crucial role in the regulation of vasodilation, blood platelet aggregation and neurotransmission in a diverse family of animals. Model nitrosyl metalloporphyrins were studied to elucidate the requirements for activation and inactivation of sGC. Fenske-Hall molecular orbital calculations in collaboration with Prof. Bruce Bursten and John Graham at Ohio State University and synthetic model nitrosyl metalloporphyrin studies by this researcher suggest that in six-coordinate nitrosyl metalloporphyrins the metal-ligand bond trans to NO becomes increasingly weaker in the series Mn, Fe, CoTPP(NO)(L), where L is either N-methylimidazole or pyridine. These data support a mechanism of NO activation whereby NO binding to the heme iron of sGC causes the scission of the proximal histidine-iron bond that may trigger a conformational change in the protein. In agreement with the proposed activation mechanism, Cu(II)PPIX and Ni(II)PPIX, obligate four-coordinate metalloporphyrins, activated heme-depleted sGC. |
