Shin-ichi OZAKI, Toshitaka MATSUI and Yoshihito WATANABE
[J. Am. Chem. Soc. 118, 9784 (1996)]
Myoglobin (Mb), normally a carrier for molecular oxygen can catalyze hydrogen peroxide supported peroxygenation of a variety of substrates, including olefin epoxidation and thioether sulfoxidation. However, the turnover numbers for sulfoxidation by Mb are lower than the values obtained by the incubation with peroxidases. Catalysis by Mb is presumably associated with an intermediate equivalent to compound I of peroxidase; however, the exact location of one of the two oxidation equivalents has not clearly been defined yet. To identify active site residues controlling ferryl oxygen transfer reactions, we have performed site-directed mutagenesis studies of sperm whale Mb. Mutating Leu29 to a histidine residue improves the rate and enantioselectivity for the oxidation of thioanisole. The rate increases versus wild type Mb are 15-fold and 22-fold for L29H and L29H/H64L Mb, respectively. On the contrary, the elimination of His64 in the distal pocket causes about 70% decrease in the oxidation rate with respect to the recombinant wild type Mb. The dominant formation of R by the mutation is in contrast with 97% ee for S enantiomer given by F41L HRP. The extremely high stereoselectivity with 97% incorporation of 18O from H218O2 into the sulfoxide for L29H/H64L Mb clearly indicates the ferryl oxygen transfer to thioanisole and rules out the involvement of molecular oxygen and hydroxyl radical. Regardless of the size and electronic properties for para substituents of thioanisole, the L29H/H64L mutant oxidizes sulfides faster than the wild type with higher enantioselectivity.