Probing the Diphosphoglycerate Binding Pocket of HbA and HbPresbyterian (β108Asn → Lys)

Abstract

HbPresbyterian (beta 108Asn --> Lys, HbP) contains an additional positive charge (per alpha beta dimer) in the middle of the central cavity and exhibits 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 functional 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 fluorescent analogues of the natural allosteric effector 2, 3-diphosphoglycerate (DPG) [Gottfried, D. S., et al. (1997) J. Biol. Chem. 272, 1571-1578]. Time-correlated single-photon counting fluorescence lifetime studies were used 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 the native and mutant proteins bind the probe at a weak binding site and a strong binding site; in all cases, the binding to HbP was stronger than to HbA. 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 also hydrolyzes p-nitrophenyl acetate, through a reversible acyl-imidazole pathway linked to the His residues of the beta beta cleft, at a considerably higher rate than does HbA. This implies a perturbation of the microenvironment 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 cavity have been transmitted to the beta beta cleft of the protein, even in its liganded conformation. This is consistent with a newly described quaternary state (B) for liganded HbPresbyterian and an associated change in the allosteric control mechanism.

Domain-Specific Effector Interactions within the Central Cavity of Human Adult Hemoglobin in Solution and in Porous Sol−Gel Matrices: Evidence for Long-Range Communication Pathways,

“…The addition of either IHP or Clto the 1:1 solution of PyTS and Hb (deoxy or CO) results in an increase in PyTS fluorescence, indicative of an increase in the relative concentration of free PyTS as it is being displaced at the DPG site by IHP. This result is consistent with the earlier proposal that the DPG binding site is the strong binding site for PyTS (38) and the recent results cited above regarding the absence of PyTS binding for G3P-Hb A.…”

“…Effect of Ligation State on Binding of PyTS to Hb A. As in a previous study (38), we have observed herein that in general PyTS binds more effectively to deoxy Hb A than to ligated Hb A, which underlies its ability to act as an allosteric effector and lower oxygen affinity. This preferential binding to the deoxy state is pH-dependent, with both ligated and unligated states showing a decrease in the level of PyTS binding with increasing pH, as expected due to partial deprotonation of residues in the DPG binding site at high pH.…”

“…Binding that does not engender such changes is not easily monitored since the effectors are often optically silent. An approach to studying effector binding that has proven to be successful utilizes fluorescent analogues of DPG ( − ). Upon binding, these analogues exhibit a large decrease in their fluorescence intensity and lifetime due to excited-state quenching by the nearby heme groups.…”

“…The use of HPTS is complicated in some instances because of the pH dependence of its fluorescence spectrum. The use of pH insensitive PyTS (1,3,6,8-pyrenetetrasulfonate) eliminates this potential complication (38). The previous studies addressed the binding of these analogue effectors to Hb A as a function of different ligation and oxidation states.…”

β93 Modified Hemoglobin: Kinetic and Conformational Consequences

“…This conclusion follows from the observation that addition of effectors the β93 derivatives does not produce the same degree of slowing of the bimolecular rebinding as is observed for HbA. An earlier study using a fluorescent analogue of DPG (73) showed that at pH values comparable to those used in the present study, the CO derivative of (PEG2000) 2 XLHbA binds the DPG analogue even more effectively than COHbA. It follows that maleimide induced alteration of β93 does not interfere with effector binding to the R-state (it may even enhance it), but does to some extent disrupt the communication pathway through which effector binding impacts R-state reactivity as reflected in the geminate and bimolecular rebinding processes.…”