Theoretical Investigation On The Catalytic Mechanisms Of Several Non-heme Iron Enzymes

Non-Heme iron enzyme is a class of iron-dependent enzyme without the porphyrin ligand.Non-Heme iron enzymes can catalyze a wide range of chemical reactions,such as electrophilic aromatic substitution,hydrogen atom abstraction,hydroxylation,monooxygenation,dioxygenation,ring closure and expansion,ring cleavage.The mechanism for dioxygen activation represents one of the core issues in metalloenzymes.Non-heme iron enzymes,which usually pocessess flexible coordination environment of iron,are challenging to study in experiments.The catalytic cycle of Non-Heme is difficult to be captured by laboratory methods because these highly reactive intermediates do not exhibit the intense spectral features characteristic of the porphyrin ligand.As such,we have carried out multi-scale simulations to decipher the activation of oxygen catalyzed by Non-Heme iron enzymes,that is particularly important to understand the mechanism of catalytic reaction regulated by protein microenvironment.In this work,molecular docking,molecular dynamics simulation(MD),quantum chemistry and molecular mechanics(QM/MM)methods were used to study the mechanism of C-C bond cleavage,hydroxylation reaction and N-nitrosation reaction catalyzed by three iron-containing oxidases.We have explored the binding mode of substrate in the active center of enzyme,the role of iron center and the key residues in the catalytic reaction.In particular,the complex spin states and electron transfer of iron during the reaction were studied.The catalytic reaction mechanisms of these iron containing oxidases have been clarified.The results can contribute to understanding the catalytic mechanism of these enzymes,which is necessary for the related experimental research and enzyme design.The main content of this thesis is summarized as below:1.Myo-inositol oxygenase(MIOX)catalyzes the oxygen activation and conversion of myo-inositol(MI)to D-glucuronic acid(GlcUA)in the absence of additional reductants.The combined molecular dynamics(MD)simulations and quantum mechanical/molecular mechanics(QM/MM)calculations have been performed to address the longstanding issue of the "dioxygen activation" by the nonheme diiron monooxygenase myo-inositol oxygenase(MIOX).MIOX utilizes a mixed-valence Fe2(Ⅲ)Fe1(Ⅱ)cluster for catalysis.It is well recognized that the Fe2(Ⅲ)site is responsible for the substrate myo-inositol(MI)binding,while the Fe1(Ⅱ)site is responsible for O2 binding and activation.However,it is enigmatic how the O-O bond of oxygen is reductively cleaved in the absence of additional reductants.In this study,we demonstrate a spin-regulated inner-sphere electron-transfer mechanism that is involved in the catalytic reactions of MIOX.Because of the Pauli principle and exchangeenhanced reactivity,the spin-regulated inner-sphere electron transfer enables the formation of an unprecedented Fe2(Ⅲ)Fel(Ⅱ)-peroxyhemiketal intermediate that is responsible for the reductive O-O cleavage,In contrast to Fe1(Ⅲ)-mediated O-O cleavage in the Fe2(Ⅱ)Fe1(Ⅲ)-peroxyhemiketal intermediate proposed previously,our calculations demonstrate that the proton transfer-triggered Fe1-O cleavage in Fe2(Ⅲ)Fe1(Ⅱ)-peroxyhemiketal intermediate is the most favorable pathway,leading to MIOOH intermediate and the Fe1(Ⅱ)species.The following Fe1(Ⅱ)-mediated O-O homolysis in MI-OOH generates the substrate radical and Fe(Ⅲ)-OH species,during which the Fel(Ⅳ)=O intermediate would be bypassed.Thus,our calculations show that both Fe sites are cooperately involved in O2 activation in MIOX and such cooperation is well regulated by the spin-dependent innersphere electron transfer.These findings of O2 activation by MIOX may have far-reaching implications on other related nonheme diiron monooxygenases.2.The SznF enzyme contains heme-oxygenase-like domain(HDO domain)utilizes a Fe2(Ⅱ)Fe1(Ⅱ)cluster catalyze selectively hydroxylation of Nω-methyl-Larginine(L-NMA)to generate Nδ-hydroxy-Nω-methyl-L-Arg(L-HMA)and Nδ,Nωdihydroxy-Nω’-methyl-L-Arg(L-DHMA),which is a key step in the synthesis of nitrosourea pharmacophore of the pancreatic cancer drug streptozotocin(SZN).Both Fe(Ⅱ)atoms are involved in the activation of oxygen,resulting in the formation of P species.In this study,we discussed several possible forms of P species and finally identified the butterfly geometry P species as the key intermediate for catalytic reactions.The conformational distribution obtained of molecular dynamics simulation explained the selectivity of hydroxylation reaction.Our study shows that the P species perform the hydroxylation of unmethylated guanidino nitrogen of L-NMA via electrophilic(2e-)attack using its σ*orbital,which sequentially affords L-HMA and L-DHMA.3.The SznF enzyme contains a mononuclear non-heme iron in cupin domain,which catalyzes the critical N-N rearrangement reaction in biosynthesis of the Nnitrosourea product,one of key donors for the functional pharmacophore.In this study,the combined molecular dynamics(MD)simulations and quantum mechanical/molecular mechanics(QM/MM)calculations have been performed to decipher the mechanism of the SznF-catalyzed N-nitrosation reaction,in which a fourelectron oxidation process between the iron center and substrate was involved.Our calculations show that the substrate L-DHMA is bound to Fe with its deptonated form and O2 binding generates Fe(Ⅲ)-O2·-species.Then,Fe(Ⅲ)-O2·-attacks onto the substrate to form the peroxo bridge intermediate,which then undergoes the homolytic cleavage of O-O bond,leading to a Fe(Ⅲ)-O·-intermediate.This is followed by the homo lytic cleavage of Cα-N1 bond and simultaneous formation of the Fe(Ⅳ)=O intermediate.The following reaction steps H-abstraction reaction by Fe(Ⅳ)=O the proton-coupled electron transfer(PCET)process were found to be facile,affording the final product L-NHMA.