Abstract:

The coordination of heme propionate with Mn²⁺ in active center of chloroperoxidase(CPO) was confirmed by means of removing the metal ions using ethylene diamine tetraacetic acid(EDTA) ligand competition reaction and determining Mn²⁺ by graphite furnace atomic absorption and inductively coupled plasmas atomic emissive spectrometry assay. The presence of Mn²⁺ was found playing a role in holding the active domain and maintaining preferential conformation of CPO based on the UV-Vis absorption spectroscopy, circular dichroism(CD) analysis as well as chlorination activity and peroxidation activity measurement before and after Mn²⁺ being removed from CPO. The maximum absorption at 398 nm of CPO, which displayed by iron(Ⅲ) porphyrin and named Soret band, decreased with a concomitant decrease of catalytic activity after Mn²⁺ in CPO being removed(MCPO). The analysis of CD spectra indicated that α-helix structure was uncoiling in MCPO. Furthermore, it was found that the changing of micro-environment around heme in CPO active site was a reversible process, and the catalytic activity may be recovered when Mn²⁺ was reintroduced, but it was a slow dynamic process that needed nearly 100 h. Moreover, the chlorination activity and peroxidation activity of CPO was enhanced compared with that of native CPO when exogenous Ag⁺ and Cr³⁺ are introduced instead of Mn²⁺. Especially, the introduction of Cr³⁺ could not only recover the peroxidation activity of MCPO but also elevate the relative activity to 106%. The chlorination activity of MCPO was also recovered and even enhanced by the introduing of Ag⁺. This process was related to the electron transfer involved in the enzymatic catalytic cycle.

Key words: Chloroperoxidase, Mn²⁺, Enzymatic performance, Chemical modification, Exogenous ion