STRUCTURAL ROLES OF CRITICAL RESIDUES IN COOPERATIVITY OF SCAPHARCA DIMERIC HEMOGLOBIN. A. Pardanani, M. Bonham, W. E. Royer,Jr., Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, MA 01605

High resolution atomic structures of the cooperative dimeric hemoglobin found in Scapharca inaequivalvis have implicated several residues in the cooperative mechanism. Of these, residues Phe 97, Thr 72 and His 101 have been mutated and subjected to functional and structural analysis in order to elucidate their effects on cooperative oxygen binding. Phe 97, whose side-chain undergoes the largest ligand-linked movement, has been hypothesized to be the key regulator of oxygen affinity. Mutation to leucine increases oxygen affinity by an order of magnitude with cooperativity diminished but not eliminated. Crystallographic analysis reveals that many aspects of ligand-linked transitions observed with wild-type are still present in F97L mutant. An important aspect of the ligand-linked structural transitions of Scapharca dimeric hemoglobin is a dramatic change in a cluster of interface water molecules upon ligation. To probe these interface water molecules, we have mutated Thr72 to Val which removes a single hydrogen bond between each subunit and the water network of the deoxygenated species. This isosteric mutation increases oxygen affinity to an even greater extent than seen with the F97L mutant. Interestingly, cooperativity is completely preserved, and may even be marginally enhanced. Structural studies have defined the only change to be loss of the single hydrogen bonded water molecule, demonstrating the importance of these water molecules in the cooperative mechanism. This proximal histidine, residue 101, provides the only direct coordination of the heme group to the polypeptide chain. Following work of Barrick on sperm whale myoglobin, we have mutated this residue to glycine, and are using exogenous imidizole to funtionally replace the lost histidine side chain. This mutation will directly test the importance of the direct link of the iron with the polypeptide chain in the cooperative mechanism. These mutations are providing novel insights into the manner by which two subunits can communicate.