Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (beta 108Asn -> Lys)

HbPresbyterian (beta 108Asn --> Lys, HbP) contains an additional positive c harge (per alpha beta dimer) in the middle of the central cavity and exhibi ts a lower oxygen affinity than wild-type HbA in the presence of chloride. However, very little is known about the molecular origins of its altered fu nctional properties. In this, study, we have focused on the beta beta cleft of the Hb tetramer. Recently, we developed an approach for quantifying the Ligand binding affinity to the beta-end of the Hb central cavity using flu orescent analogues of the natural allosteric effector 2,3-diphosphoglycerat e (DPC) [Gottfried, D. S,, et al, (1997) J, Biol, Chem. 272, 1571-1578]. Ti me-correlated single-photon counting fluorescence lifetime studies were use d to assess the binding of pyrenetetrasulfonate to both HbA and HbP in the deoxy and CO ligation states under acidic and neutral pH conditions. Both t he native and mutant proteins bind the probe at a weak binding site and a s trong binding site; in all cases, the binding to HbP was stronger than to H bA. The most striking finding was that for HbA the binding affinity varies as follows: deoxy (pH 6.35) > deoxy (pH 7.20) > CO (pH 6.35); however, the binding to HbP is independent of ligation or pH, The mutant oxy protein als o hydrolyzes p-nitrophenyl acetate, through a reversible acyl-imidazole pat hway linked to the His residues of the beta beta cleft, at a considerably h igher rate than does HbA. This implies a perturbation of the microenvironme nt of these residues at the DPG binding pocket. Structural consequences due to the presence of the new positive charge in the middle of the central ca vity have been transmitted to the beta beta cleft of the protein, even in i ts liganded conformation. This is consistent with a newly described quatern ary state (B) for liganded HbPresbyterian and an associated change in the a llosteric control mechanism.