Molecular engineering of myoglobin: the improvement of oxidation activity by replacing Phe-43 with tryptophan

The F43W and F43W/H64L myoglobin (Mb) mutants have been constructed to investigate effects of an electron rich oxidizable amino acid residue in the heme vicinity on oxidation activities of Mb. The Phe-43 --> Trp mutation increases the rate of one-electron oxidation of guaiacol by 3-4-fold; however, the peroxidase activity for F43W/H64L Mb is less than that of the F43W single mutant because the absence of histidine, a general acid-base catalyst, in the distal heme pocket suppresses compound I formation. More than 15-fold improvement versus wild-type Mb in the two-electron oxidation of thioanisole and styrene is observed with the Phe-43 --> Trp mutation. Our results indicate that Trp-43 in the mutants enhances both one- and two-electron oxidation activities (i.e., F43W Mb > wild-type Mb and F43W/H64L Mb > H64L Mb). The level of (18)O incorporation from H2(18)O2 into the epoxide product for the wild type is 31%; however, the values for F43W and F43W/H64L Mb are 75 and 73%, respectively. Thus, Trp-43 in the mutants does not appear to be utilized as a major protein radical site to form a peroxy protein radical in the oxygenation. The enhanced peroxygenase activity might be explained by the increase in the reactivity of compound I. However, the oxidative modification of F43W/H64L Mb in compound I formation with mCPBA prevents us from determining the actual reactivity of the catalytic species for the intact protein. The Lys-C achromobacter digestion of the modified F43W/H64L mutant followed by FPLC and mass analysis shows that the Trp-43-Lys-47 fragment gains a mass by 30 Da, which could correspond two oxygen atoms and loss of two protons.