Effect Of Heme Axial Ligand Mutation On Neuroglobin Peroxidase Activity

Heme protein is a metalloprotein with iron-protoporphyrin IX(heme)as a cofactor,responsible for performing important physiological functions such as oxygen carrying,electron transfer,catalysis or sensing.The molecular design based on the heme protein skeleton to obtain better active artificial metalloenzymes has long been a research hotspot.Neuroglobin(Ngb)is a new type of hemoglobin protein highly expressed in the vertebrate nervous system discovered in 2000.It has a high affinity for oxygen and can protect nerve cells against oxidative stress and inhibit the apoptosis cascade reaction.In our previous research,we discovered that after the design and introduction of additional intramolecular disulfide bonds,A15 C Ngb has extremely high thermal stability and is an ideal heme molecular design skeleton.This subject continues to design on this basis,hoping to obtain a peroxidase with high thermal stability and high catalytic activity that can actually be used to degrade organic dyes and halogenated phenols in water.In order to realize that Ngb has peroxidase activity,by referring to the structural characteristics of the active centers of decolorizing peroxidase and chloroperoxidase,we designed aspartic acid(Asp)to replace the 64-position axial ligand histidine(His),introduce a cavity above the heme that can bind hydrogen peroxide and the substrate.At the same time,Asp64 can form a catalytic acid-base pair with Lys67 to increase the activation rate of hydrogen peroxide and thus have better catalytic activity.The study found that the rationally designed A15C/H64 D Ngb has good decolorization/dehalogenation activity.Its catalytic efficiency for dye RB19 is ～30 times that of natural Vc Dy P,and its catalytic efficiency for halogenated phenol TCP is ～50 times that of natural DHP,Kcat is even 60% higher than the most active artificial dehaloperoxidase F43Y/H64 D Mb.Compared with the control mutant A15C/H64 S,the activity is ～10% higher,further verifying the success of the design strategy.This study not only clarified the relationship between the structure and function of heme protein,but also has potential bioremediation application prospects in the water environment polluted by anthraquinone dyes and halogenated phenols.