Heme substitution is an effective approach to change the function of hemoproteins. In this research, we have developed a method for the reconstitution of cytochrome P450BM3 (BM3) isolated from <i>Bacillus megaterium</i>. BM3 is a hemoprotein belonging to cytochrome P450 superfamily and is one of the most reactive P450s catalyzing biological oxidations. In our previous research, we have successfully altered the substrate specificity of BM3 by utilizing natural-substrate analogs, which we call decoy molecules, and achieved hydroxylation of non-native substrates such as benzene, cyclohexane, propane, etc. without the requirement for laborious mutagenesis. This BM3/decoy system exhibits very high catalytic activity and could be a promising biocatalyst for the hydroxylation of small organic molecules. Herein we attempted to utilize artificial metal-complexes as a substitute for heme, to increase catalytic activity and to expand reaction scope. As a conventional method, acid/organic solvent is used to remove the heme from target hemoproteins. However, this method is not compatible with every hemoprotein, leaving BM3 irreversibly denatured under these conditions prohibiting reconstitution with artificial complexes. On the other hand, a mild method for the reconstitution of hemoproteins has been developed, utilizing iron-limiting medium. In a previous study, we have successfully reconstituted P450s with metal-complexes using this method. However, this mild method was not applicable to full-length BM3, due to its structural complexity. Through several trials, we finally succeeded in the reconstitution of BM3 whilst maintaining the high catalytic activity typical of BM3. In addition, we prepared Mn-substituted BM3 by inserting the heme analog Mn-protoporphyrin IX into the heme binding site of BM3 and investigated the properties of this artificial enzyme.

Reconstitution of self-sufficient cytochrome P450 with artificial metal complexes

255th National Meeting (2018)

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