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.
Ozaki
S
S
Faculty of Education, Yamagata University, Kojirakawa, Yamagata 990-8560, Japan. yoshi@ims.ac.jp
Hara
I
I
Matsui
T
T
Watanabe
Y
Y
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002723:Chlorobenzoates / Q000378:metabolism; D004579:Electron Transport / Q000235:genetics; D006639:Histidine / Q000235:genetics; D007930:Leucine / Q000235:genetics; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000235:genetics* / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics*; D019163:Reducing Agents; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D013440:Sulfides / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics*; D014907:Whales
D002723:Chlorobenzoates; D009211:Myoglobin; D016877:Oxidants; D019163:Reducing Agents; D013440:Sulfides; D010649:Phenylalanine; D006639:Histidine; D014364:Tryptophan; C093850:methylphenylsulfide; D009195:Peroxidase; C000433:3-chloroperbenzoic acid; D007930:Leucine