The role of the distal histidine in myoglobin and haemoglobin

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Abstract
The distal E7 histidine in vertebrate myoglobins and haemoglobins has been strongly conserved during evolution and is thought to be important in fine-tuning the ligand affinities of these proteins1–8. A hydrogen bond between the Nɛ proton of the distal histidine and the second oxygen atom may stabilize O2 bound to the haem iron1–8. The proximity of the imidazole side chain to the sixth coordination position, which is required for efficient hydrogen bonding, has been postulated to inhibit sterically the binding of CO and alkyl isocyanides2–8. To test these ideas, engineered mutants of sperm whale myoglobin9 and the α- and β-subunits of human haemoglobin8,10–12 were prepared in which E7 histidine was replaced by glycine. Removal of the distal imidazole in myoglobin and the α-subunits of intact, R-state haemoglobin caused significant changes in the affinity for oxygen, carbon monoxide and methyl isocyanide; in contrast, the His-E7 to Gly substitution produced little or no effect on the rates and extents of O2, CO and methyl isocyanide binding to β-chains within R-state haemoglobin. In the β-subunit the distal histidine seems to be less significant in regulating the binding of ligands to the haem iron in the high affinity quaternary conformation. Structural differences in the oxygen binding pockets shown by X-ray crystallographic studies4,5 account for the functional differences of these proteins.