The coupled oxidations of sperm whale myoglobin (Mb) mutants are performed to examine active site residues controlling the regiospecific heme degradation, HPLC analysis of biliverdin isomers shows that L29H/H64L Mb almost exclusively gives biliverdin IX gamma, although H64L and wild-type Mb mainly afford the alpha-isomer. Relocation of the distal histidine at the 43 and 107 positions increases the amount of gamma-isomer to 44 and 22%, respectively. Interestingly, the increase in the ratio of gamma-isomer is also observed 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 between oxygen molecule bound to the heme iron and His or Trp is important in controlling the regioselectivity. The results of coupled oxidation kinetics, autoxidation kinetics, and redox potential of the Fe3+/Fe2+ couple are discussed with regard to their implications for the active site and mechanism of heme oxygenase.