Theoretical Investigations Of Catalytic Mechanism And Regulation Mechanism On Activity For Cysteine Dioxygenase And Its Model Complexes

A mononuclear non-heme iron-dependent enzyme is a kind of important metal loproteases, and it is involved in many important aerobic biochemical reactions. In nature, aerobic organisms need oxygen to provide energy and complete various metabolisms for their lives. However, the oxygen molecule in the atmosphere has a ground state with the triplet state, but the ground state of the oxidized organic substrates is generally the singlet state. This is a spin-forbidden reaction. On the one hand, this means that the spontaneous combustion of all organic compounds to produce carbon dioxide and water is a spin forbidden reaction; on the other hand, because of the spin forbidden, the reaction, which participates by O2in the triplet state, is a rather slow dynamics process. In this case, O2activation enzymes, which contain the transition metal, are used to solve this problem. These metalloproteases can catalyze and control the oxidation process of many organic materials with high selectivity, and the structure of their active site can change with different catalytic function. Understanding the catalytic mechanism and the regulation mechanism on O2activity of these metalloproteases is the hotspot and frontier of the international academic researches.Cysteine dioxygenase (CDO) is a mononuclear iron-dependent metalloprotease, which can be found in mammalian cells, especially abundant in hepatocytes. CDO plays a crucial role in the metabolism and bioconversion. CDO catalyzes the oxidation of cysteine(Cys) to cysteine sulfinate, and this is the first step for metabolizing Cys, followed by generating sulfate or taurine in the next oxidation processes. In addition, CDO plays an important role in health.CDO is a novel non-heme iron(â…¡) dioxygenase. The typical structure of the active site of non-heme iron(â…¡) dioxygenases is a2-His-1-carboxylate facial triad. However, a third histidine imidazole group takes the place of the carboxylate ligand in CDO, forming a His3facial triad. In addition, the thiol sulfur of Cys forms a thioether bond with the C of a nearby tyrosine residue. This rare Tyr-Cys adduct is termed "cross-linked protein derived cofactor". The structural properties of CDO may affect the catalytic mechanism.However, in contrast to its important role, little is known about its mechanism. The proposed reaction mechanism of CDO is currently disputed. As the enzyme reacts very fast, it is very difficult to capture the related intermediates in the reaction process by using the experimental method, and this makes it become a challenging work to further understand CDO. So, theoretical studies at the molecular level seem to be extraordinarily necessary, and carrying out the related researches is an important task for the theoretical Chemistry workers.In this paper, by performing the density functional theory calculations, we carry out the theoretical investigations of the catalytic mechanism and the regulation mechanism on activity for cysteine dioxygenase and its model complexes. It covers the CDO catalytic reaction mechanism, oxygen activation mechanism, as well as the electronic structures and charge transfer paths for CDO and its model complexes. The important and valuable results in this paper can be summarized as follows:1. DFT calculations have been carried out to investigate the catalytic mechanism of cysteine dioxygenase. The X-ray structure of rat CDO (PDB ID:3ELN)34is used as the initial structure. The first coordination sphere of iron is judged to be important to the catalytic reaction, and the three metal-bound histidine groups (His86, His88and His140), the substrate Cys, and the dioxygen molecule are included in the computational model. The geometrical structures and electronic structures of all critical points along the reaction pathways in the singlet, triplet and quintet state have been explored. And the calculated potential energy surfaces are also given. A new catalytic mechanism of rat CDO is proposed in our study, which is the "Fe-proximal oxygen" mechanism.It is found that in the mechanism, this reaction consists of five steps. Firstly, the swing of O(1)-O(2) bond between Fe and S makes Fe-proximal oxygen atom O(1) accessible to S and Fe-terminal oxygen atom O(2) accessible to Fe. This step generates a seven-membered ring intermediate INT2via transition state TS1. Then, with the bond-breaking of O(1)-O(2) bond and the intramolecular rotation of S-O(1) bond, the transfer of the second atom O(2) to the sulfoxide completes the reaction, producing the cysteine sulfinic acid.The reaction of CDO proceeds mainly on the quintet surface, and the activation energy in the rate-limiting step is15.7kcal.mol-1. The electronic characterizations are analyzed to further understand the mechanism. During the swing of O(1)-O(2) bond in the first step, an electron is transferred from Ï€*OO to σ*x2-y2.5INT2has electronic configuration Ï€*xy2Ï€*xz1Ï€*yZ1σ*z21σ*x2-y21,ad the oxidation state of Fe is â…¡. In the second step, the elongation and the bond-breaking of O(1)-O(2) bond lead to the formation of an intermediate5INT3. In this complex, the sulfur atom has radical character (ps=0.46), which is caused by the transfer of an electron from a lone-pair on sulfur to O(1) atom. At the same time, iron transfers an electron to atom O(2), and the metal changes its oxidation state from â…¡ in5INT2to â…¢ in5INT3. In the third step, with the sufficient space supplied by the previous two steps, the intramolecular rotation of S-O(1) bond produces intermediate5INT4. In the final two steps, an electron transfer takes place from the sulfoxide to the oxo-iron system, producing the cysteine sulfinic acid. And the oxidation state of Fe changes back to â…¡ in this non-heme enzyme.The "Fe-proximal oxygen" mechanism is stepwise with large exothermicity in the product formation progress. Compared with the "Fe-terminal oxygen" mechanism from de Visser’s group, it can be known that, the two mechanisms are both favorable and competitive in the catalytic process. This study gives an additional insight of the reaction mechanism of CDO.The corresponding results have been accepted in Journal of Theoretical and Computational Chemistry.2. DFT calculations have been carried out to investigate the modes for O2activation in the oxidation reaction process by CDO model complexes. The electronic structures, bonding descriptions and frontier orbitals have been analyzed, and the role of thiolate ligand and its relative position in the oxygen activation have been theoretically investigated. Four models have been selected:two of them are thiolate-ligated complexes[(iPrBIP)Feâ…¡(SPh)(Cl)](A) and [(iPrBIP)Feâ…¡(SPh)(OTf)](B), and the other two are non-thiolate-ligated complexes [(iprBIP)Feâ…¡(Cl)2](C) and [(iPrBIP)Feâ…¡(OTf)2](D). In the thiolate-ligated complexes, compared with the sixth open coordination of iron, the PhS-ligand of Model (A) is at trans position and the PhS-ligand of Model (B) is at cis position. Additionally, two key points of the O2activation process have been investigated:the first one, in which O2is not bound to the sixth open site of iron, reflects the intrinsic properties of the activation of O2, whereas the second one, in which O2is bound to that site, reflects the interactions between O2and models.It is found that the thiolate ligand is essential for the activation of O2, and its relative position plays a critical role in determining the oxidation reaction path. In the thiolate-ligated models, the PhS-ligand can simultaneously function as the Ï€-donor and the σ-donor to transfer charge to Feâ…¡ via an operative interaction. On the one hand, this decreases the effective nuclear charges of Feâ…¡ and lowers the redox potentials; on the other hand, the good electron-donating ability reduces the sixth ligand binding energy of the oxygen molecule by decreasing the structure Lewis acidity. The PhS-ligand provides a prerequisite for the activation of O2. The relative position of PhS-ligand also plays an important role in O2activation process. When PhS-ligand coordinates at the trans position, the Fe d-based MO will be the highest occupied molecular orbital and transfers charge to the O(1)2p-based MO as the σ-donor. It will lead to the oxidization of Fe and the Fe-S bond will break. When PhS-ligand coordinates at the cis position, the S Ï€-based MO are the highest-energy double occupied molecular orbitals and S transfers charge to the O(2) as the σ-donor. It will lead to the oxidization of S in PhS-ligand.Combined with the experimental datas, the redox potentials for the four (iPrBIP)Feâ…¡ complexes and the amount of charge transfer are correlated. Interestingly, the amount of charge transfer of PhS-ligand is closely correlated with the related redox potentials. The more charge it transfers, the lower the related redox potentials are. It indicates that we may improve the model through adjustment of charge transfer ability of the ligand.This study provides a theoretical basis for further understanding the oxygen activation mechanism of CDO. The corresponding results have been published in J. Phys. Chem. A (2012,116,5510-5517).3. DFT calculations have been carried out to investigate the CDO model complexes, the active site of CDO and their corresponding selenium-substituted complexes. In the first two steps of the O2activation reaction, the bonding descriptions, frontier orbitals and charge transfer paths have been analyzed. Considering the electron donating ability, Se is stronger than S, why the CDO cannot catalyze the oxidation of Sec but that of Cys? It is still an open question. In order to solve it, the theoretical research has been carried out. Two models have been selected: one is the CDO model complex and the other one is the active site of CDO (PDB ID:3ELN). According to the reported CDO catalytic mechanism in the literatures, the shared structures A, B and C in the first two steps have been explored. They are the structure A, in which O2is not bound to the sixth open site of iron; the structure B, in which O2is just bound to that site; and the structure C, which is a four-membered ring structure, producing by the attack of the proximal oxygen atom on the sulfur of CysIt is found that in the first reaction step during the O2binding process, Se in the Sec-bound complexes has the stronger electron-donor ability to transfer charge to Fe in comparison with S. Take as an example the CDO model complexes, in the structure A, the spin-up electron is singly occupied in the four Fe d-orbital, and the oxidation state of Fe is â…¡. When O2is coordinated, Fe transfers the charge of d orbital into the OOÏ€*-based MO in the structure B, and the oxidation state of Fe becomes â…¢. This structure is consistent with the characteristics of the ferric-superoxo species. But, in Se-CDO model complexes, Fe also transfers the charge of d orbital into the OOÏ€*-based MO. Because of the better charge transfer ability of Se, Se can compensate the Fe center for the loss of electron by transferring the charge of the Se4p-based MO to the Fe d-based MO. So, the oxidation state of Fe keeps â…¡ in structure B.In the second reaction step, during the four-membered ring structure formation process, S has a better electron-donating ability than Se. With the formation of the four-membered ring structure, the S(Se)-O(2) bond is generated, and Fe-O(1) bond is weakened. In the structure C of CDO model complexes, on the one hand, S transfers the charge of the S3p-based MO into the OOÏ€*-based MO, pairing up with one electron in the OOÏ€*-based MO; on the other hand, S can transfer the charge into the Fe d-orbital. During this process, the oxidation state of Fe changes back to â…¡ And in the structure C of Se-CDO model complexes, the Fe-Se bond is weakened more greatly than the Fe-S bond, and Se can only transfer the charge of the Se4p-based MO into the OOÏ€*-based MO, without transferring charge to Fe. And Fe remains the â…¡ oxidation state throughout the reaction.Be similar with the model complexes, in the active site of CDO, the change of the oxidation state for the Fe center is â…¡â†'â…¢â†'â…¡ But, for the active site of Se-CDO, the oxidation state of the Fe center is always keeping â…¡.During the first two reaction steps for the Cys-bound complexes and the Sec-bound complexes, the competition of the charge transfer between S and Se causes the different change of the oxidation state for the Fe center. Considering that the ferric-superoxo species is an active oxidant in such reaction, the Fe center in the Cys-bound complexes can effectively catalyze the O(1)-O(2) bond cleavage, while the Fe center in the Sec-bound complexes cannot. These results are helpful to understand why CDO cannot catalyze the oxidation of Sec but the oxidation of Cys.The corresponding results have been submitted in Journal of Inorganic Biochemistry.