Mutation of residue Phe⁹⁷ to Leu disrupts the central allosteric pathway in Scapharca dimeric hemoglobin

Residue Phe⁹⁷, which is thought to play a central role in the cooperative functioning of Scapharca dimeric hemoglobin, has been mutated to leucine to test its proposed role in mediating cooperative oxygen binding. This results in an 8-fold increase in oxygen affinity and a marked decrease in cooperativity. Kinetic measurements of ligand binding to the Leu⁹⁷ mutant suggest an altered unliganded (deoxy) state, which has been confirmed by high resolution crystal structures in the unliganded and carbon monoxide- liganded states. Analysis of the structures at allosteric end points reveals them to be remarkably similar to the corresponding wild-type structures, with differences confined to the disposition of residue 97 side chain, F-helix geometry, and the interface water structure. Increased oxygen affinity results from the absence of the Phe⁹⁷ side chain, whose tight packing in the heme pocket of the deoxy state normally restricts the heme from assuming a high affinity conformation. The absence of the Phe⁹⁷ side chain is also associated with diminished cooperativity, since Leu⁹⁷ packs in the heme pocket in both states. Residual cooperativity appears to be coupled with observed structural transitions and suggests that parallel pathways for communication exist in Scapharca dimeric hemoglobin.