Blocking the Gate to Ligand Entry in Human Hemoglobin

His(E7) to Trp replacements in HbA lead to markedly biphasic bimolecular CO rebinding after laser photolysis. For isolated mutant subunits, the fraction of fast phase increases with increasing [CO], suggesting a competition between binding to an open conformation with an empty E7 channel and relaxation to blocked or closed, slowly reacting states. The rate of conformational relaxation of the open state is similar to 18,000 s(-1) in alpha subunits and similar to 10-fold faster in beta subunits, similar to 175,000 s(-1). Crystal structures were determined for tetrameric alpha(WT)beta(Trp-63) HbCO, alpha(Trp-58)beta(WT) deoxyHb, and Trp-64 deoxy-and CO-Mb as controls. In Trp-63(E7) beta CO, the indole side chain is located in the solvent interface, blocking entry into the E7 channel. Similar blocked Trp-64(E7) conformations are observed in the mutant Mb crystal structures. In Trp-58(E7) deoxy-alpha subunits, the indole side chain fills both the channel and the distal pocket, forming a completely closed state. The bimolecular rate constant for CO binding, k'(CO), to the open conformations of both mutant Hb subunits is similar to 80-90 mu M-1 s(-1), whereas k'(CO) for the completely closed states is 1000-fold slower, similar to 0.08 mu M-1 s(-1). A transient intermediate with k'(CO) approximate to 0.7 mu M-1 s(-1) is observed after photolysis of Trp-63(E7) beta CO subunits and indicates that the indole ring blocks the entrance to the E7 channel, as observed in the crystal structures of Trp(E7) deoxyMb and beta CO subunits. Thus, either blocking or completely filling the E7 channel dramatically slows bimolecular binding, providing strong evidence that the E7 channel is the major pathway (>= 90%) for ligand entry in human hemoglobin.