Control of heme reactivity by diffusion: Structural basis and functional characterization in hemoglobin mutants

The effect of mutagenesis on O-2,CO, and NO binding to mutants of human hem oglobin, designed to modify some features of the reactivity that hinder use of hemoglobin solutions as blood substitute, has been extensively investig ated. The kinetics may be interpreted in the framework of the Monod-Wyman-C hangeux two-state allosteric model, based on the high-resolution crystallog raphic structures of the mutants and taking, into account the control of he me reactivity by the distal side mutations. The mutations involve residues at topological position B10 and E7, i.e., Leu (B10) to Tyr and His (E7) to Gln, on either the a chains alone (yielding the hybrid tetramer Hb alpha (Y Q)), the chains alone (hybrid tetramer Hb (YQ)), or both types of chains (H b(YQ)). Our data indicate that the two mutations affect ligand diffusion in to the pocket, leading to proteins with low affinity for O-2 and CO, and es pecially with reduced reactivity toward NO, a difficult goal to achieve. Th e observed kinetic heterogeneity between the alpha (YQ) and beta (YQ) chain s in Hb(YQ) has been rationalized on the basis of the three-dimensional str ucture of the active site. Furthemore, we report for the first time an expe riment of partial CO binding, selective for the chains, to high salt crysta ls of the mutant Hb(YQ) in the T-state; these crystal lo graph ic data may be interpreted as "snapshots" of the initial events possibly occurring on l igand binding to the T-allosteric state of this peculiar mutant Hb.