Theoretical Study Of Catalytic Reaction Mechanisms Of Iron-Containing Enzymes

Iron is the most abundant trace element in most living organisms.Iron-containing enzymes have a wide range of biological functions and they are involved in important physiological processes,such as oxygen storage and transport,electron transfer,oxygen activation,and biomolecular metabolism.Iron-containing enzymes are divided into heme iron enzymes,non-heme iron enzymes,and iron-sulfur cluster enzymes.Studying the reaction mechanism of iron-containing metalloenzymes is of great importance to understanding the life process,material metabolism,and the design of biomimetic catalysts.Due to the complexity of the iron-containing enzymes’structure and the high efficiency of the enzyme-catalyzed reaction,the reactive intermediates are very difficult to capture by experiment method,thus it is very difficult to explain the reaction mechanism by experimental means alone.In this dissertation,quantum chemical cluster approach and quantum mechanics/molecular mechanics（QM/MM）approach are used to study the catalytic reaction mechanism of nonheme,binuclear nonheme,iron-sulfur cluster enzymes,and molybdenum-iron nitrogenase.The details are as follows:（1）Quantum chemical cluster approach was performed to elucidate and delineate the reaction mechanism of a nonheme iron enzyme Egt B,which catalyzes the C-S bond and S-O bond formation betweenγ-glutamyl cysteine（γGC）and N-α-trimethyl histidine（TMH）in the ergothioneine biosynthesis.Two different mechanisms were considered,depending on whether the sulfoxidation or the S-C bond formation takes place first.The calculations suggest that the S-O bond formation occurs first between the thiolate and the ferric superoxide,followed by homolytic O-O bond cleavage.Subsequently,proton transfer from a second-shell residue Tyr377 to the newly generated iron-oxo moiety takes place,which is followed by proton transfer from the TMH imidazole to Tyr377,facilitated by two crystallographically observed water molecules.Next,the S-C bond is formed betweenγGC and TMH,followed by proton transfer from the imidazole CH moiety to Tyr377,which was calculated to be the rate-limiting step for the whole reaction,with a barrier of 17.9 kcal/mol in the quintet state.The calculated barrier for the rate-limiting step agrees quite well with experimental kinetic data.Finally,this proton is transferred back to the imidazole nitrogen to form the product.The alternative thiyl radical attack mechanism has a very high barrier,being 25.8 kcal/mol,ruling out this possibility.（2）Aur F is a diiron enzyme that utilizes two dioxygen molecules as the oxidant to catalyze the oxidation of p-aminobenzoate to p-nitrobenzoate.Quantum chemical cluster approach was performed to elucidate the reaction mechanism of this enzyme.Two different models were considered,with the oxygenated intermediate being a diferric peroxo species or a diferric hydroperoxo species.The calculations strongly favor the model with a diferric peroxo species.The reaction starts with the binding of a dioxygen molecule to the diferrous center to generate aμ-η²:η² diferric peroxide complex.This is followed by the cleavage of the O-O bond,concertedly with the formation of the first N-O bond,which has a barrier of only 9.2 kcal/mol.The oxidation of the p-hydroxylaminobenzoate intermediate requires the binding of a second dioxygen molecule to the diferrous center to generate the diferric peroxide complex.Similar to the oxidation of p-aminoben-zoate,the O-O bond cleavage and the formation of the second N-O bond take place in a concerted step.The p-nitrobenzoate product is formed after the release of two protons and two electrons from the substrate.Theμ-η¹:η¹ hydroperoxo species give a much high energy barrier of 28.7 kcal/mol for the substrate oxidation due to the large energy penalty for the generation of the active hydroperoxo species.（3）[4Fe-4S]containing（R）-2-hydroxyisocaproyl-Co A dehydratase catalyzes the dehydration of（R）-2-hydroxyisocaproyl-Co A in the fermentation of L-leucine.QM/MM calculations were performed to elucidate the mechanism and the stereoselectivity of the enzyme.Six possible anti-ferromagnetically spin couplings（ααββ,αβαβ,αββα,ββαα,βαβα,βααβ）were considered for the syn-and anti-elimination.The first step is the ligand substitution,the substrate binds to the iron-sulfur with theα-hydroxy group,with the best spin state ofααββ.After the Ser37 residue forms a hydrogen bond with the carbonyl group of the substrate,one electron is transferred from[4Fe-4S]⁺to the substrate,a ketyl radical anion intermediate is formed.Next,it’s the heterolytic cleavage of the C_α-O bond,a Fe-OH^-group is formed.Subsequently,a syn-elimination takes place,the Fe-OH^-group abstracts the C_β-hydrogen of the substrate as a proton,generating the experimentally identified allylic ketyl radical intermediate,with a barrier of 10.7 kcal/mol,which is the rate-limiting step for the whole reaction.The anti-elimination to get the Z-isomer was also considered,the calculation showed that the anti-elimination is both energetically and thermodynamically unfeasible,which is consistent with the experimental observations.The work here should be helpful for the understanding of the mechanism of other enzymes of this family.（4）Quantum chemical cluster approach was performed to elucidate the mechanism of the dissociation of a sulfur ligand ions of molybdenum-iron nitrogenase.Starting from A₀(Mo^III4Fe^III3Fe^II),the reaction undergoes four proton-coupled electron transfer steps to form the A₄ intermediate(Mo^III2Fe^III5Fe^II),in which S1B,S2B,and S3A are protonated with one hydride binds to Fe4 and Fe5.The S3AH^-ligand ion abstracts a proton from S1BH^-,and the S3A ligand ion dissociates from the Fe Mo cluster in the form of H₂S,with an energy barrier of 7.2 kcal/mol.After another proton-coupled electron transfer step,the E₄intermediate(Mo^III7Fe^II)is formed,in which S1A,S2B,and S5A are protonated with two hydrides bridging two iron ions（Fe1-Fe2,Fe1-Fe4）and the carbide is protonated.This E₄intermediate is consistent with the experimental spectral results.Subsequently,the E₄intermediate undergoes a reduction elimination reaction of two hydride ions to generate H₂,forming a highly reducing Mo^III5Fe^II2Fe^I,and the reaction is only endergonic by 0.9kcal/mol.N₂ coordinates with one of the Fe^I ions（Fe4）,and the reaction is endergonic by1.4 kcal/mol.Therefore,it is only endergonic by 2.3 kcal/mol for the dissociation of H₂ and the coordination of N₂ refers to E₄.This result is in line with the experimental observations.This study provides theoretical support for the dissociation mechanism of sulfur ligand ions on the molybdenum-iron nitrogenase.