Effects of the arrangement of a distal catalytic residue on regioselectivity and reactivity in the coupled oxidation of sperm whale myoglobin mutants

The coupled oxidations of sperm whale myoglobin (Mb) mutants are performed to examine active site residues controlling the regiospecific heme degradat ion, HPLC analysis of biliverdin isomers shows that L29H/H64L Mb almost exc lusively gives biliverdin IX gamma, although H64L and wild-type Mb mainly a fford the alpha-isomer. Relocation of the distal histidine at the 43 and 10 7 positions increases the amount of gamma-isomer to 44 and 22%, respectivel y. Interestingly, the increase in the ratio of gamma-isomer is also observe d by a single replacement of either His-64 with Asp or Phe-43 with Trp. It appears that the polarity of the active site as well as hydrogen bonding be tween oxygen molecule bound to the heme iron and His or Trp is important in controlling the regioselectivity. The results of coupled oxidation kinetic s, autoxidation kinetics, and redox potential of the Fe3+/Fe2+ couple are d iscussed with regard to their implications for the active site and mechanis m of heme oxygenase.