Distal Histidine In The Heme Active Site Manipulatrs The Peroxidase Of Myoglobin

The stability and correct folding of a protein are the basis for it to implementdifferent biological functions, therefore, the study of protein structure and functionhas always been the hot spot of life scientific research. Myoglobin (Mb) is one of themost studied proteins.Generally, by mutating the amino acid in Mb or by modifying it’s heme activecenter, the researcher can study the relationship between the structure and the function.In order to further study the effect of amino acid on the structure and function of Mb,and to understand the structure and the function relationship of Mb, in this work, weexpressd and purified the Mb mutants in E.coli, then we investigated their structureand function by using UV-visible spectroscopy etc.Through the preparation of competent E.coli, cells transformation, vaccination,expanding culture, inducing expression to express Mb mutants efficiently, and thenthrough cells harvest, sonication, ultrafiltration, anion exchange chromatography,ultrafiltration, gel filtration chromatography or dialysis, we could obtain purified Mbmutants, L29H Mb, where Leucine29(Leu, L) was mutated to histidine (His, H), andmutant L29H/F43H Mb, where both Leucine29and Phenylalanine43(Phe, F) aremutated to histidine.By experimental study on L29H Mb, this work reveals the role of double distalhistidines (His29/His64) in the catalytic function of Mb. The results showed that thetwo histidines in L29H Mb can cooperatively oxidizie guaiacol, and it has aperoxidase activity3.7-fold higher than that of WT Mb(wild-type Mb, WT Mb).Moreover, as L29H/F43H Mb mutant has three distal hisitidines formed ametal-binding site，this work studied the peroxidase activity of L29H/F43H Mb andthe peroxidase activity after it bound with Cu (II) or Zn (II). The results showed that the peroxidase activity of L29H/F43H Mb can be inhibited to a different extent bybonding of Cu (II) or Zn (II), which provides valuable information for the lowperoxidase activity observed for native cytochrome c oxidase.This paper provides new insight into the structure and function relationship forMb, as well as new idea and new method for the rational heme protein design.