9405492 Ackers Previous work under this grant achieved a breakthrough in this problem by developing strategies that resolve the functional energetics of hemoglobin tetramers in all ten states of hemesite ligation, using non-oxygen ligands. This has opened the door to obtalning previously-inaccessible knowledge regarding properties of the intermediate species and their roles in the cooperative mechanism. Work using the 2 analog cyanomet indicates that: (a) the hemoglobin molecule acts as a three-state molecular switch to control ligand affinity; (b) the intermediate state has a quaternary T structure and has stored energy through ligand-induced tertiary structure changes; (c) this free energy of "tertiary constraint" pays for the early stage of cooperativity prior to the quaternary T~R switch; (d) the T structure can accommodate tertiary constraint in only one dimeric half-molecule, leading to a Symmetry Rule for the quaternary transition: T~R switching occurs when each dimeric halfmolecule acquires ligated hemesites. In current work, we are extending these studies to mutant hemoglobins in order to map the structural pathways of free energy transduction and to establish the connections with oxygen binding. We shall use multiple combinations of altered amino acid residues and ligated hemesites to determine contrlbutions of local residue energies to the quaternary T to R switch. Parallel studies will test the correspondence between cyanomet ligation and oxygenation. This research will use a combination of techniques, including quantitative low temperature isoelectric focusing and high precision binding of oxygen. The results from this research will serve as a model forstudying "molecular codes" in other complex regulatory protein assemblies. %%% The goal of this research program is to determine the molecular mechanism of cooperative oxygenation in human hemoglobin. We are attempting to decipher the rules and energetic driving forces whereby the ten molecular for ms (each with a structurally-unique combination of ligated and unligated heme sites) respond to the interactions with heme-site ligands and with small regulatory molecules including protons, chloride ions, and 2,3- diphosphoglycerate. ***
