Abstract:

Methanobactin(Mb) is a copper-binding small peptide that appears to function as an agent for Cu sequestration and uptake in methanotrophs. It can coordinate a single Cu ion by its nitrogens from two oxazo-lone rings and its sulfurs from two enethiol groups. Mb-Cu is possibly in direct association with the particulate methane monooxygenase(pMMO) and plays a more direct role in pMMO loading and activity. It has been reported that Mb-Cu can increase electron flow to the activity centers of pMMO and exhibit redox activity. This suggested that Mb may be used to mimic oxidoreductase. In this paper, to explore the potential peroxidase ca-talytic properties in this novel class of Cu binding compound, Mb from Methylosinus trichosporium IMV3011 was isolated and purified by HP20, supelco LC-C₁₈ and IDE-Cu immobilized metal-chelate affinity chromatography column. Chrome azurol S(CAS) calorimetric assays showed that the Mb have high Cu(II) affinities. Analysis of the Mb-Cu sample by LC/TOF MS gave a peak at m/z 1215, corresponding to an m/z 1153 Mb molecule which has lost one or two hydrogen atoms and bound one copper atom. Fluorescent and UV-Vis spectroscopy results also demonstrated that the transitions occurring when Cu(II) interacts with Mb. These results indicated that Mb has been loaded with Cu ion and Mb-Cu has been prepared. Using Mb-Cu as a mimic of peroxidase, the kinetics of oxidative reaction of hydroquinone with hydrogen peroxide catalyzed by Mb-Cu were investigated by UV-Vis spectrophotometer. The change of hydroquinone concentration was determined by monitoring the decrease of absorbance(A) at 288 nm. The apparent first order rate constants(K_obs) show that the reaction rate for the catalytic oxidation of hydroquinone increases by a factor of 4×10² for the Mb-Cu at 25 ℃. The mechanism of hydroquinone oxidation catalyzed by Mb-Cu was proposed, and a kinetic model was established. The effect of temperature, Mb-Cu addition amount and concentration of hydrogen peroxide on the cata-lysis reaction were investigated. The results show that Mb-Cu exhibite the general characteristics of the bioca-talyst but had higher thermal stability than that of natural peroxidase. K_obs was found to be enhanced as tempe-rature increased from 25 ℃ to 60 ℃. Therefore, Mb-Cu can be used as an effective mimetic peroxidase in the process of hydroquinone oxidation.

Key words: Methanotroph, Methanobactin, Hydrogen peroxide, Hydroquinone, Kinetics, Mimetic peroxidase