A Novel Method For Determining Mn AOS And Studies On The Characteristics Of Mn(Ⅱ)/Mn(Ⅲ) Oxidation By Purified Recombinant CueO In Vitro

Manganese(Mn)oxide is a kind of highly reactive mineral,which is widely found in the deep sea,soil and sediment environments.Accurate determination of Mn AOS is a key step to understand the structures,properties,environment behaviors and potential applications of Mn oxides.The traditional methods for determining Mn AOS have the disadvantages of complicated operation,long analysis period and little applicability to the micro samples,and each method has some limitations.The bacterial Mn(Ⅱ)oxidation reaction is an extremely complex process.The catalytic oxidation of multicopper oxidases is considered to be the main reason for the formation of manganese oxides.However,the catalytic oxidation mechanism is not very clear,and it is urgent to explore characteristics of Mn(Ⅱ)/Mn(Ⅲ)oxidation in vitro by the recombinant protein CueO,of which manganese oxide gene were cloned and purified.The discovery of this mechanism,can help us to better understand the biological mineralization process for multicopper oxidases catalyzing.It plays an important role in the regulation of its oxidation behavior,the migration process of heavy metals and organic pollutants and the process of geochemical cycle!In this study,a novel two step colorimetric method was developed to determine the Mn AOS of vernadite,acid birnessite and hausmannite,and the oxalic acid-permanganate back titration method,and X-ray absorption near-edge spectroscopy(XANES)were adopted to determine the Mn AOS of the three Mn oxides for comparison.Moreover,we investigated the characteristics and the electron transfer of Mn(Ⅱ)/Mn(Ⅲ)oxidation in vitro by the recombinant protein CueO,which was cloned from E.coli K-12 MG1655 and purified.In the system of low concentration manganese and copper ions,with the help of DFOB to capture Mn(Ⅲ),the content of Mn(Ⅲ)-DFOB and high manganese with the reactant of Mn(Ⅱ)/Mn(Ⅲ)were monitored regularly.The main results are as follows:1.The novel two step colorimetric method,was combined with leucoberbelin blue I(LBB)and formaldoxime colorimetric method which were employed to obtain the content of high-valent Mn and total Mn,respectively.This novel method was used determine the Mn AOS of vernadite,acid birnessite and hausmannite,which was 4.00 ± 0.02、2.67 ± 0.01 and 4.00 ± 0.01,respectively.The result obtained by this method is more accurate than that obtained by the oxalic acid-permanganate back titration method and the XANES method.In addition,this novel method is also appropriate for trace amount of samples(0.005 g).2.The recombinant protein CueO can oxidize Mn(Ⅱ)to manganese oxides in the system with low concentration of manganese and copper ions(2 mM MnCl2,0.4 mM CuCl2),to eliminate the effect of chemical oxidation,to further confirm the manganese oxidizing activity of CueO protein in vitro.At the same time,the manganese oxidation activity of CueO protein was significantly inhibited with Na4P2O7 solution,and the inhibition was increased with the concentration increasing.When adding high concentration of Na4P2O7(5 mM),the manganese oxidation activity of CueO protein could be completely inhibited.3.In the study of the characteristics of Mn(Ⅱ)oxidation by recombinant protein CueO in vitro,the content of Mn(Ⅲ)-DFOB and high manganese with the reactant of Mn(Ⅱ)were monitored regularly in the system.The experimental results show that CueO protein can oxidize Mn(Ⅱ)to Mn(Ⅲ),with the oxidation efficiency reaching 50%,and the addition of DFOB could enhance Mn(Ⅱ)oxidation by CueO protein.At the same time,the color of the solution was not changed after the regular sampling mixed with LBB,which indicates that this experiment could not create Mn(Ⅳ).4.In the study of the characteristics of Mn(Ⅲ)oxidation by recombinant protein CueO in vitro,the content of Mn(Ⅲ)-DFOB and high manganese with the reactant of Mn(Ⅲ)acetate dihydrate(C6H9MnO6)were monitored regularly in the system.The results showed that the addition of CueO could inhibit the reduction of Mn(Ⅲ).At the same time,the color was not changed after the sample mixed with LBB,indicating that the CueO protein could not oxidize Mn(Ⅲ)to Mn(Ⅳ),so the CueO protein could not catalyze the two-step electron transfer of the manganese oxidation process alone.