Formation and catalytic roles of compound I in the hydrogen peroxide- dependent oxidations by His64 myoglobin mutants

A His64 → Asp mutant of sperm whale myoglobin (Mb), H64D Mb, has been prepared to mimic the active site of chloroperoxidase from the marine fungus Caldariomyces fumago, in which distal glutamic acid is suggested to enhance compound I formation by H₂O₂. The H64D mutant allows us to see the accumulation of compound I in the reaction of Mb with H₂O₂ for the first time. The successful observation of compound I is due to at least 50-fold improvement in the formation rate of compound I as well as its stabilization upon the His64 → Asp replacement. Catalytic activity of wild-type Mb and a series of His64 Mb mutants (H64A, H64S, H64L, and H64D Mb) are examined for one-electron oxidation and oxygenation by using H₂O₂ as an oxidant. The H64D mutant is the best catalyst among the myoglobins and shows 50-70-fold and 600-800-fold higher activity than the wild type in the one-electron oxidations and peroxygenations, respectively. The origin of the varied activity upon the mutations is discussed on the basis of the formation rate and stability of compound I.