Myoglobin (Mb) has been one of the most intensively investigated hemoproteins as evident from the accumulated biochemical, biophysical, and spectroscopic data. 1 The heterologous overexpression system for recombinant sperm whale Mb in Escherichia coli has been developed, 2 and high-resolution X-ray crystal structures of the wild type as well as some Mb mutants are available. 3 Thus, superposition of the active site structures of Mb and other hemoproteins enables us to utilize Mb as heme enzyme models for the elucidation of structurefunction relationships. 4 We have engineered sperm whale Mb based on the comparison of crystal structures of oxymyoglobin and an oxy form of cytochrome c peroxidase (CcP)(Figure 1). 5 Although the Leu-29 f His and His-64 f Leu double replacement of Mb seems to create a peroxidase-like active site, the imidazole is located too far from the heme center to interact with hydroperoxide bound to the iron. Our previous results suggest that L29H/H64L Mb cannot efficiently cleave OO bond to generate a ferryl (FeIV dO) radical cation species, equivalent to compound I of peroxidase. 6 Thus, we have mutated Phe-43 to a histidine residue because the predicted distance between His-43 and the heme iron is approximately equal to that of CcP. The novel F43H/H64L Mb mutant oxidizes sulfide and styrene more efficiently than peroxidase. More intriguingly, we have identified a compound I-like species of the Mb mutant as the catalytic intermediate for the first time. The replacement of Phe-43 in the wild type with a histidine residue increases the rate of thioanisole oxidation by 14-fold, and the mutation of His-64 f Leu in F43H Mb further enhances the sulfoxidation rate by 13-fold (Table 1). The enantiomeric excess is improved from 25% to 85% by the His-64 f Leu and Phe-43 f His double mutation of Mb, and the dominant enantiomer is R. More than 92% of 18O incorporation in the sulfoxide from H2 18O2 in the oxidation by wild type, F43H, and F43H/H64L Mb indicates that the ferryl oxygen is transferred to thioether. In comparison with the wild type, the F43H/H64L mutant oxidizes styrene 300 times faster with an improvement of enantioselectivity from 9 to 68%. Incubations of styrene and H2 18O2 with wild type and F43H/H64L Mb resulted in incorporation of 20% and 94% of 18O in epoxide, respectively. The low 18O incorporation into the epoxide in the presence of the wild type and H2 18O2 could be rationalized by the competition of the ferryl oxygen transfer and cooxidation mechanism. The cooxidation mechanism requires protein radical formation followed by binding of molecular oxygen to generate a protein-peroxy radical, and His-64 was suggested as the initial radical site. 7 The replacement of His-64 with an unoxidizable leucine residue could prevent generation of the protein radical and decrease the cooxidation. In fact, the value of 18O incorporation from 18O-labeled hydrogen peroxide for the F43H mutant, bearing two histidines in the active site, is 54%, which is between the values for F43H/H64L and wild type Mb. 8

We have attempted to identify the catalytic species of F43H/H64L Mb involved in a net two-electron oxidation of thioanisole and styrene. The horseradish peroxidase compound I-like spectrum is not observed by monitoring the changes in absorption spectra of the incubation mixture containing the mutant and hydrogen peroxide. 9 However, the mixing of F43H/H64L Mb and m-chloroperbenzoic acid (mCPBA) causes the decrease in absorbance at 406 nm, followed by the shift to longer wavelength by 12 nm (Figure 2a). The formation of the first intermediate proceeds at the rate of kobs1) 110 s-1 (standard …