REACTION OF VARIANT SPERM-WHALE MYOGLOBINS WITH HYDROGEN-PEROXIDE - THE EFFECTS OF MUTATING A HISTIDINE RESIDUE IN THE HEME DISTAL POCKET

The reaction of hydrogen peroxide with a number of variants of sperm-w hale myoglobin in which the distal pocket histidine residue (His(64)) had been mutated was studied with a combination of stopped-flow spectr oscopy and freeze-quench EPR. The rate of the initial bimolecular reac tion with hydrogen peroxide in all the proteins studied was found to d epend on the polarity of the amino acid side chain at position 64. In wild-type myoglobin there were no significant optical changes subseque nt to this reaction, suggesting the rapid formation of the well-charac terized oxyferryl species. This conclusion was supported by freeze-que nch EPR data, which were consistent with the pattern of reactivity pre viously reported [King and Winfield (1963) J. Biol. Chem. 238, 1520-15 28]. In those myoglobins bearing a mutation at position 64, the initia l bimolecular reaction with hydrogen peroxide yielded an intermediate species that subsequently decayed via a second hydrogen peroxide-depen dent step leading to modification or destruction of the haem, In the m utant His(64) --> Gln the calculated electronic absorption spectrum of the intermediate was not that of an oxyferryl species but seemed to b e that of a low-spin ferric haem, Freeze-quench EPR studies of this mu tant and the apolar mutant (His(64) --> Val) revealed the accumulation of a novel intermediate after the first hydrogen peroxide-dependent r eaction. The unusual EPR characteristics of this species are provision ally assigned to a low-spin ferric haem with bound peroxide as the dis tal ligand. These results are interpreted in terms of a reaction schem e in which the polarity of the distal pocket governs the rate of bindi ng of hydrogen peroxide to the haem iron and the residue at position 6 4 governs both the rate of heterolytic oxygen scission and the stabili ty of the oxyferryl product.