Studies On Relative Reaction Mechanisms During The Catalytic Process Of Class ?a Ribonucleotide Reductase

Ribonucleotide reductase?RNR?can catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides,which is a key and rate-limiting enzyme for gene duplication,mutation and repair.Its activity is closely related to the formation and metastasis of cancer cells.Therefore,it's an important target for cancer treatment and screening of anticancer drugs.The studies on the structure and the catalyzing mechanisms of the enzyme are crucial important to the discovery of anticancer drugs for human beings.Class ?a RNR??aRNR?is a trimer composed of two homodimeric subunits designated R1 and R2?or?2 and?2?.A reversible radical tranfer pathway of ?aRNR consists of a series of conserved aromatic amino acids have been found,Tyr122??[Trp48?]??Tyr356??Tyr731??Tyr730??Cys439?,in which Tyrl22O·is oxidized by the activity diiron center.This pathway enables radical to deliver from Tyrl22O·on the?subunit to Cys439 on the?subunit through about a 35?distance,which then initiates the transformation from ribonucleotides to deoxyribonucleotides.However,the questions about the possible proton/electron transfer reactions between the Tyr356O·and Tyr731,the formation of the active Tyr122O·-Fe?III?Fe?III?cluster and the composition of the active intermediate X in the long-range radical-tansfer pathway have not been clarified at the molecular level today.In this paper,using density functional theory?DFT?and ONIOM method,the electron transfer mechanisms during the ?aRNR catalytic process were deeply explored.Three main works have been done,which are summarized as follows:?1?Three models for the active site structure of intermediate X in class ?a ribonucleotide reductase have been studied by using broken-symmetry-DFT approach and spin-unrestricted Kohn-Sham-DFT solution.The DFT methods are carried out to optimize the models of the intermediate X to find the suitable methods for non-heme enzyme with diiron center.By comparing the calculated data of geometries and spin densities for the X model clusters with available experimental and theoretical data,the performances of 12 density functional theory?DFT?methods on intermediate X calculations have been systematically assessed for the first time in this work.BP86,BVP86 and M06L are uniformly recommended.?2?Using M06-BS/GENECP method and considering the amino acids near the diiron center,such as Asp84,Asp237,Trp48 and H118,This chapter examined the rationality structure of X and clarified the detail of electron transfer from Tyr122 to the diiron center,which explains the functionality of the diiron cluster of ?aRNR in the long-range proton-coupled electron transfer reactions.The DFT calculated results show that if the initiate structure of intermediate X is H₂O-Fe₁?III?-??-O?₂-Fe₂?IV?,the generation of Tyr122O·requires two step reactions.The first step is a single-proton transfer reaction and the second step is a proton coupled electron transfer reaction.If the second oxygen atom?O₂?of double oxygen bridge ligands[??-O?₂]in the diiron center is unprotonated,the hole are always remained at the diiron center regardless of proton transfer from Tyr122 to the OH ligand of diiron.These results indicate that electron transfer can take place from the side chain of Tyr122 to the Fe?IV?site on the diiron center only after the protonation of O₂ in bridge ligands.The OH ligand of Fe?III?functions as the proton acceptor to accept the proton of Tyr122,which promotes the generation of the first amino acid radical for the long-range electron hole transfer of ?aRNR.This is consistent with the reported experimental results.?3?Possible interaction conformations between?and?subunits were simulated by molecular dynamics.The electron/proton transfer from Tyr731 to Tyr356O·was studied by considering the insertions of Glu350 and water molecules?H₂Os?between the side chains of Tyr356O·and Tyr731.DFT calculations reveals that the proton/electron transfer reactions between the Tyr356O·and Tyr731 can be achieved by a variety of connecting ways.When the side chains of Tyr356O·and Tyr731 interact directly with a hydrogen bond,the proton/electron transfer reactions take place through a typical proton-coupled electron transfer?PCET?mechanism.When several H₂Os connect two tyrosine side chains via hydrogen bonds,the reactions occur via a double-proton coupled electron transfer?dPCET?mechanism for a water bridge and a triplet-proton coupled electron transfer?tPCET?mechanism for two water bridges.When the side chain of Glu350 links Tyr356O·with Tyr731,the corresponding reactions take place through the dPCET mechanism with a lower energy barrier,indicating that the side chain of Glu350 can promote proton/electron transfer from Tyr731 to Tyr356O·.For the case of both H₂Os and the side chain of Glu350 inserting between the side chains of Tyr356O·and Tyr731 simultaneously,the proton/electron transfer reactions occur through the tPCET?a H₂O and Glu350?or a quartet-proton coupled electron transfer?qPCET,two H₂Os and Glu350?mechanism.Interestingly,there are two cases for the participation of Glu350,an oxygen atom and two oxygen atoms of the carboxyl group with the similar energy barriers.If considering zero-point vibrational energy,in these cases,some energy barriers are less than 0,which indicates that the connecting hydrogen bonds are low-barrier hydrogen bonds and proton transfer can be carried out without barriers.In general,the forward energy barriers for the proton/electron transfer reactions from Tyr731 to Tyr356O·through direct hydrogen bonding,several H₂Os,the sides chain of Glu350,and both H₂Os and Glu350 are all low,indicating that the reactions between Tyr356O·and Tyr731 can easily occur as long as forming the effective proton connection channels.Glu350 and H₂Os not only are the bridges of proton transfer between the electron donor and acceptor,but also play a vital role in stabilizing the donor and acceptor when proton and electron are synchronously delivered through different paths.