The Importance Of Asn52 In The Structure-Property-Catalytic Function Of Human Cyt C

Heme protein is a kind of metalloprotein containing protoporphyrin IX(heme)cofactor.How the same heme binds to different protein molecular frameworks and performs various physiological functions,that is,exploring the structure-function relationship of heme protein has always been the focus of protein research.As an evolutionarily conserved heme protein,Cytochrome c containing heme c co-moieties and plays an essential role in the survival of organisms by participating in the electron transport of the mitochondrial respiratory chain and inducing endogenous apoptosis.Homologous sequence alignment revealed the Asparagine52(Asn52)of Cytochrome c is a highly conserved site that may be involved in the electron transfer process and form a cardiac phospholipid binding pocket.However,in recent years,large-scale gene sequencing analysis has found that a small number of people will appear N52 S mutation,its biological significance needs to be further clarified.In this subject,we characterized the structural and catalytic properties of N52 S Cyt c and N52 A Cyt c mutants by a series of biochemical and spectroscopic approaches.The circular dichroism spectrum(CD)shows that both N52 S and N52 A mutations have a significant effect on the secondary structure of the protein,which shows that Asn52 plays an important role in the stability of protein structure.EPR experiments show that N52 S Cyt c have both high and low spin states under near physiological conditions,while N52 A Cyt c and WT Cyt c only have low spin states.This indicates that after the Asn52 mutation was Ser52,the microenvironment around heme has changed greatly.Subsequently,the coordination competition experiments of sodium azide show that the exchange rate of axial ligand Met80 in N52 S Cyt c is three times faster than WT Cyt c,further indicating that mutations in Asn52 sites affect the coordination state of iron porphyrin.Through the above experiments,we found that the Met80 coordination ability in N52 S Cyt c is weaker than WT.The ron porphyrin.is easier to bind hydrogen peroxidase because of the dissociation of the Met80.Therefore,it can be inferred that N52 S Cyt c has higher peroxidase activity than WT.For this,we used UV-vis spectra and stopped-flow to carry out heme degradation experiments and peroxidase kinetics experiments on different substrates.The data confirmed that the peroxidase activity of N52 S Cyt c increased by 8 times and ?1.3 times higher than that of WT Cyt c and N52 A Cyt c,respectively.This suggests that Asn52 is a site that inhibits Cyt c peroxidase activity and may reduce the occurrence of accidental apoptosis.This study further clarifies the contribution of highly conserved Asn52 residues in Cyt c,and provide a deeper understanding of the relationship between structure-property-function of Cytochrome c...