Activation of the low oxygen affinity-inducing potential of the Asn108(beta)-> Lys mutation of Hb-Presbyterian on intramolecular alpha alpha-fumaryl cross-bridging

The Asn108 beta-->Lys mutation in hemoglobin (Hb-Presbyterian mutation) end ows a low O-2 affinity-inducing propensity to the protein. Introduction of a fumaryl crossbridge between its two alpha 99 lysine residues also induces a low O-2 affinity into HbA. We have now engineered an alpha alpha -fumary l cross-bridge into Hb-Presbyterian to determine the synergy or additivity, if any, that can be achieved between these two low O-2 affinity-inducing s tructural perturbations. Despite the presence of the additional epsilon -am ino group of Lys108(beta) within the central cavity, the epsilon -amino gro up of Lys99(alpha) of deoxy Hb-Presbyterian retained high selectivity for a lpha alpha -fumaryl cross-bridging, with an overall efficiency comparable t o that with HbA, The alpha alpha -fumaryl cross-linking of Hb-Presbyterian reduced its O-2 affinity much more significantly than that observed with Hb A, indicating a synergy between the two low O-2 affinity-inducing structura l perturbations. Apparently, the alpha alpha -fumaryl cross-bridge in Hb-Pr esbyterian activates part of the latent low O-2 affinity-inducing potential of Lys108(beta) that is generally activated in the presence of chloride. T he synergy between the Asn108(beta)-->Lys mutation and the alpha alpha -fum aryl cross-bridging was conserved in the presence of chloride, but not in t he presence of DPG, Furthermore, in the presence of chloride and DPG, alpha alpha -fumaryl Hb-Presbyterian accessed a low O-2 affinity T-state that is accessed by HbA, alpha alpha -HbA and Hb-Presbyterian only in the presence of IHP, Isoelectric focusing analysis suggested that the alpha alpha -fuma ryl cross-linking of Hb-Presbyterian induces changes in the ionization beha vior of one or more of the functional groups neighboring Lys99(a) and Lys10 8(beta) [presumably His103(alpha) and/or Glu101(beta)] to compensate for th e extra positive charge of Lys108(beta), Molecular modeling studies identif ied two potential chloride binding sites per alpha beta dimer within the mi ddle of the central cavity of alpha alpha -fumaryl HbA involving residues H is103(alpha), Arg104(beta) and Asn108(beta), The affinity of these sites is increased in alpha alpha -fumaryl Hb-Presbyterian as a result of the Asn10 8(beta)-->Lys mutation. Thus, the results of the present study suggest that the enhanced neutralization of the positive charges in the middle of the c entral cavity of Hb achieved by these two electrostatic modifications, one (the alpha alpha -fumaryl cross-bridge) acting directly and the other (the Presbyterian mutation) acting indirectly through the mediation of chloride ion binding, facilitates the alpha alpha -fumaryl-Hb Presbyterian to access a low O-2 affinity T-state structure much more readily than either Hb-Pres byterian or alpha alpha -fumaryl HbA.