title: Hybridization of modified-heme reconstitution and distal histidine mutation to functionalize sperm whale myoglobin.
abstract: To modulate the physiological function of a hemoprotein, most approaches have been demonstrated by site-directed mutagenesis. Replacement of the native heme with an artificial prosthetic group is another way to modify a hemoprotein. However, an alternate method, mutation or heme reconstitution, does not always demonstrate sufficient improvement compared with the native heme enzyme. In the present study, to convert a simple oxygen storage hemoprotein, myoglobin, into an active peroxidase, we applied both methods at the same time. The native heme of myoglobin was replaced with a chemically modified heme 2 having two aromatic rings at the heme-propionate termini. The constructed myoglobins were examined for 2-methoxyphenol (guaiacol) oxidation in the presence of H2O2. Compared with native myoglobin, rMb(H64D.2) showed a 430-fold higher kcat/Km value, which is significantly higher than that of cytochrome c peroxidase and only 3-fold less than that of horseradish peroxidase. In addition, myoglobin-catalyzed degradation of bisphenol A was examined by HPLC analysis. The rMb(H64D.2) showed drastic acceleration (>35-fold) of bisphenol A degradation compared with the native myoglobin. In this system, a highly oxidized heme reactive species is smoothly generated and a substrate is effectively bound in the heme pocket, while native myoglobin only reversibly binds dioxygen. The present results indicate that the combination of a modified-heme reconstitution and an amino acid mutation should offer interesting perspectives toward developing a useful biomolecule catalyst from a hemoprotein.
authors.affiliation: PRESTO, Japan Science and Technology Agency, Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Sato
authors.forename: Takashi
authors.initials: T
authors.lastname: Hayashi
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Ando
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Hisaeda
authors.forename: Takafumi
authors.initials: T
authors.lastname: Ueno
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Watanabe
mesh_terms: D000818:Animals; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D009211:Myoglobin / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D009195:Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014907:Whales
chemical_list: D009211:Myoglobin; D006418:Heme; D006639:Histidine; D009195:Peroxidase