Theoretical Studies On Biosynthesis Mechanisms Of Tryptophan Tryptophyl Quinone In PreMADH And Electron Relay Function Of Midway Tryptophan

The proton/electron transfer in proteins participates in many biological enzyme-catalyzed reactions,and even the key steps or speed-determining steps of many enzyme-catalyzed reactions,and plays a vital role in many life activities.Therefore,studying the related proton/electron transfer reaction mechanism in the process of enzyme catalysis is important to understand the catalytic mechanism of many biological enzymes,and also provides important information for exploring the mystery of life activities.MADH exists in Gram Negative Bacteria and Parococcus denitrificans.It can catalyze the oxidation of methylamine to form formaldehyde and ammonia,so that these bacteria can use methylamine as the only source of carbon,nitrogen and energy.Therefore,studying the biosynthesis mechanism of the MADH prosthetic group and learning how organisms use amine compounds as energy sources are of great significance for mankind to expand energy sources.The synthesis process of TTQ involves the transfer reaction of 6 proton-electron,but the details of these 6proton-electron transfer reactions have not been reported so far.Therefore,this paper mainly uses the ONIOM calculation method to study the proton/electron transfer process of Pre MADH through 6-electron transfer reaction to generate TTQ to carry out the work,it is specifically divided into the following two aspects:（1）In the process of PreMADH losing 6 electrons to generate TTQ,it can be divided into three independent reactions.The three reactions are cross-link formation betweenβTrp OH57 andβTrp108,insertion of a hydroxyl group at C6 ofβTrp OH57,and the quinol is oxidized to quinone.In this reaction,each step loses two electrons and is accompanied by the transfer of protons.This paper aims to use the method of ONIOM（B3LYP-D3/6-31+G（7）d,p（8）:PM6）to calculate and analyze the three-step reaction ofβTrp OH57 andβTrp108 to generate TTQ in pre MADH.Studies have found that the energy barrier of the single radical process is smaller than the energy barrier of the double radical process,which is more conducive to the Mau G to catalyze the generation of MADH from pre MADH to synthesize TTQ.The first two-step two-electron oxidation reaction involves the C-H bond cleavage reaction on the indole ring.The energy barrier that needs to be overcome is higher,the reaction speed is slower,and the time required is longer,which is consistent with the experimental results.According to the potential energy analysis of the entire reaction,whether it is through a single radical process or a double radical process,the C-H cleavage step at the C6 site ofβTrp OH57 is the decisive step of the entire reaction.In each step of the two-electron oxidation process,Asp32 and Asp76 close to the reaction center act as proton acceptors,and play an important role in the process of proton transfer from the active center to the surroundings.The H₂O in the protein will build a proton transfer bridge to promote the transfer of protons from the active center to Asp32 and Asp76,thereby ensuring the effective progress of catalysis.（2）In order to investigate the distribution of the electron density and the corresponding reaction potential energy of the cross-reaction of βTrpOH57 andβTrp108with Trp199 participation,and verify Trp199 plays an essential electron transfer function in the six-electron transfer process.The third chapter of the paper uses the DFT method to calculate the details of the six electron transfer with the participation of Trp199.The result shows that Trp199 participates in the six-electron oxidation reaction of Pre MADH to generate TTQ,only the first two-electron oxidation reaction has a spin density distribution on Trp199.This also shows that Trp199 participates in the electron transfer in this process,it is oxidized to Trp199 radical,this shows that the protonation state of Trp199 regulates the 6-electron transfer of Trp199 in the process of TTQ synthesis.And the two electron transfer reactions in the second and third steps show that there is no electron distribution on Trp199,and may not participate in the reaction.Among them,the energy barrier of the forward reaction in the first electron transfer process is 29.8Kcal/mol,which is the decisive step of the entire reaction.The study found that the energy barrier of the decisive step when Trp199 did not participate in the reaction was smaller than that of the decisive step when Trp199 participated in the reaction,indicating that the reaction was more difficult to proceed with the participation of Trp199.In summary,Trp199 plays an important role as an electron transfer channel in the six-electron long-range transfer reaction of Pre MADH to TTQ.It provides ideas for further study of electron transfer in proteins.