Molecular Simulation Study On The Mechanism Of Radical Generation And Transfer In Ribonucleotide Reductaseand The Mechanism Of Co-Activation Of Galactose Oxidase By Hydroxyl Radicalsand O₂

Ribonucleotide reductase（RNR）catalyzes the reduction of ribonucleotides（NDPs/NTPs）to form corresponding deoxyribonucleotid-es（d NDPs/d NTPs）,providing the required precursors for DNA synthesis and repair.Therefore,the study of RNR is helpful to understand the essence of life.Class Id and Ie RNR are newly discovered RNR subclasses,theirβsubunits are different from the previously discovered subclasses in structure and function and use different radical initiators to start the radical transfer.Class Ie RNR uses DOPA radical（DOPA·）as the radical initiator,however,the mechanism of DOPA·generation,transfer and regeneration is still unclear.Class Id RNR employs the dimanganese cofactor in the superoxidized state(Mn^III/Mn^IV)as the radical initiator,however,the structure of Mn^III/Mn^IV and the mechanism of radical initiation and transfer are still unclear.It is necessary to reveal the mechanisms of radical generation,initiation,transfer and regeneration of the two subclasses,as it is one of the core contents of RNR research and can be helpful for the control of pathogens and the development of anti-pathogen drugs.Our laboratory research group immobilized galactose oxidase（GO）by coordinating copper（Cu（II））and zirconium（Zr（IV））with galactose oxidase in PBS.Cu（II）and Zr（IV）cooperative catalyze the decomposition of by-product H₂O₂ to produce hydroxyl radical·OH and dioxygen O₂.It was found that the catalytic efficiency of the immobilized galactose oxidase was greatly improved.The mechanism of activating galactose oxidase needs to be further studied and clarified.In this paper,the mechanism of radical generation,initiation,transfer and regeneration in class Ie RNR and Id RNR and the mechanism of co-efficient activation of galactose oxygenase by·OH and O₂ were studied through molecular dynamic（MD）simulations,quantum mechanical/molecularmechanical（QM/MM）calculations and density functional theory（DFT）.The main contents are as follows.（1）Mechanism of DOPA·generation,transfer and regeneration in class Ie RNR.Based on DFT simulations,first,the mechanism of three-electrons oxidation of Tyr126 to DOPA·was studied.The superoxide O₂^·-provided by the flavoprotein Nrd I cannot directly oxidize the residue Tyr126 to DOPA·.It should be protonated to hydroperoxyl radical HO₂^·.The calculation results suggest that the covalent modification of Tyr126 and the DOPA·generation can be carried out with no involvement of metal cofactors.Then,the mechanism on the transfer of DOPA·between DOPA126 and Trp52 was studied.The calculations have demonstrated that Lys213 is a key residue for the DOPA·transfer.Theε-amino group of Lys213 is used not only as a bridge for the electron transfer but also as a proton donor.It can provide a proton to DOPA126 via a water molecule,and thus the radical transfer from DOPA126 to Trp52 is facilitated.It has also been revealed that the protonation of Asp88 is the prerequisite for the DOPA·generation andtransfer in class Ie RNR.Finally,the mechanism of radical regeneration was studied.Once the radical is quenched,it can be regenerated via the oxidations by O₂^·-and HO₂^·.（2）Mechanism of radical initiation and transfer in class Id RNR.Based on the simulations of DFT and electron paramagnetic resonance（EPR）,first,we determined that the Mn^III-（μ-oxo）₂-Mn^IV（complex 2）and Mn^III-（μ-hydroxo/μ-oxo）-Mn^IV（complex 1）clusters are the structures of the dimanganese cofactor for class Id RNR in the superoxidized state（active）.Complex 1 is the monoprotonated state of complex 2.Then,based on the determined dimanganese cofactors,the mechanism of radical initiation and transfer in class Id RNR is revealed.The Mn^III-（μ-oxo）₂-Mn^IV cluster in complex 2 has not enough reduction potential to initiate radical transfer,instead,it needs to be monoprotonated into Mn^III-（μ-hydroxo/μ-oxo）-Mn^IV（complex 1）before the radical transfer.The protonation state ofμ-oxo can be regulated by changing the protein microenvironment,which is induced by the aggregation and separation ofβsubunit withαsubunit.The radical transfer between the Mn^III-（μ-hydroxo/μ-oxo）-Mn^IV cluster and Trp30 is a water mediated tri-proton-coupled electron transfer（t PCET）,which transfers proton from theε-amino group of Lys71 to the carboxylate group of Glu97 via W551 and the bridgingμ-hydroxo ligandthrough a three-step reaction.Thisnewlydiscovered proton-coupled electron transfer mechanism in class Id RNR is different from those reported in the known Ia-Ic RNRs.Theε-amino group of Lys71,which serves as a proton donor,plays an important role in the radical transfer.（3）Mechanism of cooperative and efficient activation of GO by·OH and O₂.Based on DFT simulations,first,the mechanismof GO activation by·OH was studied.·OH can oxidize the reduced ECu（I）/（Cys-Tyr）of the active site to the active ECu（II）/（Cys-Tyr）·,and oxidize the intermediate ECu（II）/（Cys-Tyr）（inactive）to the active ECu（II）/（Cys-Tyr）·.The calculation results demonstrate that·OH（as single-electron oxidant）can activate GO by restoring the active ECu（II）/（Cys-Tyr）·in the active site.Based on DFT simulations,the mechanism of O₂（as two-electron oxidant）oxidize the reduced ECu（I）/（Cys-Tyr）of the active site to the active ECu（II）/（Cys-Tyr）·was studied.During oxidation,O₂ is reduced to H₂O₂.It has been found that the·OH activation is highly associated with the O₂reduction half-reaction(O₂-to-H₂O₂-to-（·OH+O₂）.