The Action Mechanism Study Of Mitochondrial Respiratory Chain Complex Ⅲ Active Site

Mitochondrial respiratory chain mainly depends on electron transport to complete a series of redox reactions in order to maintain normal life activities.Cytochrome bc1 complex(Complex Ⅲ)plays an important role in mitochondrial respiratory chain.Complex Ⅲ,as one of the most important fungcide targets,has two active sites of the different action mechanism:the quinone reduction(Qi)site and the quinol oxidation site(Qo).At present,the number of commercialized fungicides at the Qo site is up to twenty,while there are only three commercialized inhibitors at the Qi site.For Qi active site,the mechanism of its inhibitors is unclear at present.Through literature research,we have learned about the reduction mechanism of substrate,in which electron transfer and proton transfer occur,which involves the protonation structural differences of the three key residues around the Qi site(i.e.Lys251,Asp252,His217,the residue number is based on the mitochondrial complex Ⅲ number of the bacterial sepcies;the same position corresponds to the residue number of the bovine species Lys227,Asp228,His201).Here,it is proposed that there are four different protonation states of amino-acid residues at the Qi site,which are Lyn-NH2-Ash-COOH-Hid(o-NH),Lys-NH3-Asp-COO--Hip(double-NH),Lyn-NH2-Ash-COOH-Hie(m-NH)and Lys-NH3-Asp-COO--Hie(m-NH).Based on this,we analyzed the four protonation states by the methods of molecular simulation,molecular docking,molecular dynamics simulation and MM/PBSA.The mechanism of reported highly active Qi site inhibitors has been determined,this site inhibitors could stably bind to the surrounding residues corresponding to Qi site.The results showed that the four inhibitors of antimycin A,NQNO,Ascochlorin and GSK932121 are stable in the residue state Lyn-NH2-Ash-COOH-Hie(m-NH)at the Qi site,correspondingly.Only inhibitor GW844520 tends to exist in the residue state Lyn-NH2-Ash-COOH-Hid(o-NH).Based on this,we also analyzed and obtained the binding states of three commercialized inhibitors i.e.cyazofamid,amisulbrom and Fenpicoxamid,and a series of compounds modified by amisulbrom at the Qi site,these inhibitors exist in the residue state Lyn-NH2-Ash-COOH-Hie(m-NH).This work provides a theoretical basis for the follow-up design of reasonable inhibitors.For Qo active site,the resistance of its classical strobilurin inhibitors was highly serious.How to overcome the resistance of this kind of inhibitors was an urgent problem.The design of new inhibitors based on natural products is one of the effective ways to overcome the existing resistance.At present,the research of Qo site inhibitors has been very mature,action mode of which is known and stable.The Qo site inhibitors maintain an important hydrogen bond with the residue Glu272,and have better hydrophobic interaction with the surrounding residues.It would provide a theoretical basis for screening and designing inhibitors.Literature research showed that(+)-Neopeltolide is a new inhibitor of complex Ⅲ.Previous studies have shown that(+)-Neopeltolide belongs to Qo site inhibitor.Our team has modified its structure and obtained a series of new Qo site inhibitors.On this basis,the structure-activity relationship of these compounds was analyzed.The results showed that the terminal diphenyl ether with electron-withdrawing group could improve the activity of these compounds.This conclusion has certain guiding significance for screening and designing new inhibitors at the Qo site of complexⅢ in the future.