Theoretical Study On The Reaction Mechanism Of The Oxidation Of P-iodophenol By Horseradish Peroxidase

Horseradish peroxidase(HRP)-luminol-H2O2 chemiluminescent(CL)system with p-substituted phenol derivatives as the enhancer has been commonly used in the areas of food safety,environmental monitor and immunoassay etc.Completely understanding the enhancer-HRP-luminol-H2O2 CL reaction is not only the basis for improving the performance of this CL system,but also the precondition of increasing its application value.However,at present the precise mechanism of the HRP-luminol-H2O2-enhancer(p-substituted phenol derivatives)CL system is not clear,due to the limitation of the experimental equipment and the complexity of the CL reaction.Previous studies show that one key factor contributing to the enhancement effect of the enhancer on the luminol CL system is the reaction of HRP intermediates(HRP compound I and HRP compound II)with the enhancer.This reaction accelerates the enzyme turnover.There are many kinds of p-substituted phenol derivatives enhancers,among them,p-iodophenol(PIP)is the most commonly used enhancer.For the reaction of PIP with HRP,PIP is first oxidized by HRP compound ?(HRP-I)to generate HRP compound II(HRP-II)and PIP radical.After this,the HRP-?,as a potent oxidant,reacts with another PIP,generating the resting state HRP and the PIP radical.Using quantum chemical method,this project investigated this key factor of the enhanced HRP-catalyzed luminol CL system at atomic level.This is to trace the transition state and intermediate which are difficult measuring in experiment.This theoretical study contributes to further understanding the reaction process between enhancer and HRP intermediates,as well as helps to explain the enhancing effect of PIP on the luminol CL system.This work selects PIP as the model enhancer.Then the active pocket cluster model is built based on the active site of the HRP,some vital residues,and the substrate PIP.Finally,using quantum mechanics(QM)simulation,the mechanism of PIP oxidation by HRP is explored.The main calculating results are summarized in the following.(1)This project performs the theoretical study on the PIP oxidation by HRP-I.The substrate and HRP-I has two binding modes,which lead to two different reaction routes(path A and path B).Path A contains two successive reaction steps.The initial step is a proton-coupled electron-transfer process.The PIP loses a proton to His42 by a water molecule and offers an electron to the porphyrin cation radical.Then,the second step is a proton transfer event whereby the protonated His42 relays a proton to the iron-oxo group by a water molecule.Path B is a novel reaction route proposed in this study and it contains a single reaction step.The reactant can get to the final product via only one transition state.This step involves a proton transfer from PIP to iron-oxo group and a commitment electron from the substrate to the ferryl moiety.The final product complex led by both paths includes a HRP-II stable isomer and a PIP radical.The two reaction paths share some common features:two-state reactivity;the bridging water determines the ability of HRP-? to oxidize the PIP substrate;in terms of reaction kinetics,the energy barriers of path A and B are 2.0/2.3 kcal/mol and 0.5/0.5 kcal/mol,respectively.It indicates that both reaction routes are energetically favorable.(2)The theoretical study on the PIP oxidation by HRP-II is performed.Here too,the substrate and HRP has two binding modes,leading to two different reaction routes(path C and path D).Path C and path D are both novel reactions proposed in this study and consist of a single step.Both reaction routes are a proton-coupled electron-transfer process.The bridging water molecule abstracts a proton from PIP and loses a proton to the iron-hydroxyl moiety of HRP-?;correspondingly an electron from PIP transfers to the porphyrin cation radical of HRP-II.The product complex obtained by both paths consists of a resting state HRP and a PIP radical.Both reaction routes share some common features:a novel fresh PIP instead of the original PIP radical in the product of HRP-? will cause the elongation of iron-hydroxyl bond in HRP-II;two-state reactivity;the bridging water molecule determines the function of HRP-? to oxidize the PIP;in view of the reaction kinetics,the energy barriers of path C and D are respectively-0.3/-0.5 kcal/mol and 0.7/0.5 kcal/mol,indicating that both reaction routes are energetically favorable.This project proposed the reaction mechanism of PIP oxidation by HRP using quantum chemistry method.Our work will provide a new direction and technique means to find out the precise mechanism of the PIP enhanced luminol CL system.