Development of heme-based highly efficient biocatalysts

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As a research intended to construct heme-based highly efficient biocatalyst, we have developed a functional expression system of bovine/human hybrid cytochrome oxidase in human HeLa cells by integrating the bovine mitochondrial DNA derived subunit I gene into the cell genome, whose product is the largest with 12 trans membrane helices in the 13 different enzyme-subunits and essential to the function. The expression and subsequent import of subunit I into the inner membrane of mitochondria permit the association with the other 12 human subunits leading to the hybrid enzyme having the same dioxygen reduction and proton pumping activities as those of the human enzyme, In this system, the hybrid dominates over the human enzyme. By mutating the subunit I, we have found the key residue for the proton pumping and also demonstrated that the proton pumping can be uncoupled from the dioxygen reduction. The methods we have developed ale applicable to cure mitochondrial diseases caused by mal-func … More tion of mitochondrial DNA derived membrane proteins. We have also developed the cell-free functional synthesis of Paracoccus denitrificans cytochrome oxidase by exogenously adding the prosthetic group into E. coli reaction system.The prosthetic group, protoheme, of myoglobin (Mb) can be chemically modified to functionalize the protein as monooxygenase, peroxygenase and peroxidase. Incorporation of unnatural heme, metalloporphycene, into Mb turned out to enhance the oxygen affinity to 1000-fold. Protein engineering of Mb with the above porphycene powers up the oxidizing activity enormously to allow the oxidative destruction of bisphenol A, a notable endocrine disrupter. These results revealed that unnatural heme including chemically modified are powerful to develop a protein-based efficient catalyst.Our in vivo and in vitro functional expression system of cytochrome oxidase pave the way to produce a highly efficient biocatalyst from highly hydrophobic membrane proteins by combining the protein engineering with an incorporation of unnatural heme into the active center. Less