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 inve stigate effects of an electron rich oxidizable amino acid residue in the he me vicinity on oxidation activities of Mb. The Phe-43 --> Trp mutation incr eases 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 sing le mutant because the absence of histidine, a general acid-base catalyst, i n the distal heme pocket suppresses compound I formation. More than 15-fold improvement versus wild-type Mb in the two-electron oxidation of thioaniso le and styrene is observed with the Phe-43 --> Trp mutation. Our results in dicate that Trp-43 in the mutants enhances both one- and two-electron oxida tion activities (i.e., F43W Mb > wild-type Mb and F43W/ H64L Mb > H64L Mb). The level of O-18 incorporation from (H2O2)-O-18 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 b e 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 mod ification of F43W/H64L Mb in compound I formation with mCPBA prevents us fr om determining the actual reactivity of the catalytic species for the intac t protein. The Lys-C achromobacter digestion of the modified F43W/H64L muta nt 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.