Altered ligand rebinding kinetics due to distal-side effects in hemoglobinChico (Lys(beta 66)(E10)-> Thr)

Hb Chico is an unusual human hemoglobin variant that has lowered oxygen aff inity, but unaltered cooperativity and anion sensitivity. Previous studies showed these features to be associated with distal-side heme pocket alterat ions that confer increased structural rigidity on the molecule and that inc rease water content in the beta-chain heme pocket. We report here that the extent of nanosecond geminate rebinding of oxygen to the variant and its is olated beta-chains is appreciably decreased. Structural alterations in this variant decrease its oxygen recombination rates without significantly alte ring rates of migration out of the heme pocket. Data analysis indicates tha t one or more barriers that impede rebinding of oxygen from docking sites i n the heme pocket are increased, with less consequence for CO rebinding. Re sonance Raman spectra show no significant alterations in spectral regions s ensitive to interactions between the heme iron and the proximal histidine r esidue, confirming that the functional differences in the variant are due t o distal-side heme pocket alterations. These effects are discussed in the c ontext of a schematic representation of heme pocket wells and barriers that could aid the design of novel hemoglobins with altered ligand affinity wit hout loss of the normal allosteric responses that facilitate unloading of o xygen to respiring tissues.