Engineering of Cytochrome c: Characterization and Applications of Microperoxidases


Wen-Yih Jeng (鄭文義), Yuh-Dar Chang, and Woei-Jer Chuang

Department of Biochemistry, National Cheng Kung University Medical College, Tainan 701, Taiwan


Hemoproteins occur widely in higher organisms and carry out very diverse functions. They all contain the heme prosthetic group and can be categorized in terms of their reactions with oxygen. These proteins are responsible for hydrogen peroxide utilization and decomposition, oxygen transport and storage, electron transport, oxygen reduction, and hydrocarbon oxidation. In order to explore the diverse functions of hemoproteins, microperoxidases (MPs) are used to mimic the active site of hemoproteins. MPs are water-soluble heme species that have been used as models for the hemoproteins, and these MPs with various peptides (MP-5, MP-6, MP-8, MP-9, & MP-11) were obtained by digestion of cytochrome c with different proteases. In this study, we showed that MP catalyzed catalase-type reaction in addition to peroxidase-type and cytochrome P450-type reactions. The relative catalytic specificities of Ac-MP-8 with a histidyl ligand exhibit the following orders: peroixdase-type > cytochrome P450-type > catalase-type reactions. The fifth ligands of native peroxidase, cytochrome P450 and catalase are histidine, cysteine and tyrosine, respectively. Therefore, we expressed the cytochrome c in E. Coli to mutate the fifth ligands of MPs to minic the effects of proteins on the functions of hemoproteins. The expression of cytochrome c was achieved by constructing the genes of iso-1-cytochrome c and heme lyase from Saccaromyces cerevisiae in parallel into vector pET-21b. In addition, the H23Y, H23C, H23R, L20K, Q21K and L20RQ21R mutants of cytochrome c have been obtained to mimic the active site of hemoproteins. The His-Ac-MP-11, Cys-Ac-MP-11, and Tyr-Ac-MP-11 will be prepared from these mutants and served as the models for peroxidase, catalase, and cytochrome P450, respectively. Effects of fifth ligands on the peroxidase, catalase, and monooxygenase activities of these heme models will provide us the structure-function relationships of heme enzymes and elucidate the diverse functions of hemoproteins.