Spectra And Density Functional Theoretical Studies Of The Structures Of Bi-metallothionein, Pb-metallothionein And As-metallothionein

Metallothionein (MT) is a class of metal protein characterized by a high cysteines content (up to30%of the amino acid residues), low molar mass and lack of aromatic amino acid residues. Chemical and spectroscopic studies have shown that MT can bind a wide range of metal ions. MT can lower heavy metals toxicology, capture harmful oxidant radicals and alkylating agent. MT also plays a central role in the pharmacology of metal-containing medicines. Due to its unique physiological functions, MT has been a hot topic in the life science. Elucidating the structure of MT is important for understanding its physiological functions. Here, the structures of three kinds of MT (Bi-MT, Pb-MT and As-MT) were investigated using spectroscopy and theoretical calculation methods. The main results were shown in the following:(1) The molecular geometry, vibration frequencies and electric absorption spectrum of metal thiolate complexes M(SC6H5)3(M=Bi, Sb or As) have been calculated with density functional (mPW1PW, B3LYP, TPSSh and OLYP) and ab initio method (HF), respectively. The theoretical results were compared to the available experimental data. It is demonstrated that mPW1PW functional is superior to other functionals (B3LYP, TPSSh and OLYP) and ab initio method (HF) in predicting molecular structures and vibration frequencies of Sb-thiolate and As-thiolate complexes. For Bi-thiolate complex, the hybrid density functional B3LYP gives better performance than other methods. The theoretical electric absorption spectra of Bi(SC6H5)3from B3LYP functional is similar to the available experimental data, which confirms the rationality of theoretical calculation method. The confirmed optimal theoretical calculation method for each complex will be helpful for our further understanding on the complexation structures of apo-MT binding Bi3+,Pb2+and As3+(2) The coordination of Bi3+to rabbit liver metal-free metallothionein was investigated using both experimental and theoretical methods. Spectral results suggest that one apo-MT can bind7Bi3+ forming the product Bi7-MT. Circular dichroism spectra indicate that Bi-MT2has a different secondary structure from those of Zn-MT2and Cd-MT2. Three possible Bi3+coordination structures (BiS3O1(1), BiS3O1(2) and BiS3O1(3)) and their relative binding free energies in Bi7-MT2were calculated using the density functional theory by active-site modes. Absorption spectra corresponding to these coordination structures were evaluated by time-dependent density functional theory. By comparison of calculated results and experimental spectra, the coordination mode in Bi7-MT2was confirmed to be BiS3O1(1), and the carboxyl group in the aspartic acid residues contributes to the Bi-O bond formation. Furthermore, the theoretical method was successfully used to predict the formation process of Bi-O bond in Bi7-metallothionein, an important product in vivo on effectively reducing side effect of metal-containing drugs.(3) The binding of apo-MT, its individual domains (apo-αMT, and apo-βMT) and Zn7-MT with Pb2+at different pH conditions have been studied by UV-vis and CD spectra. For apo-MT, the product Pb7-MT was obtained from apo-MT under pH5.4-7.0, while the product Pb7-MT’ was formed under pH5.4-4.0. The spectra results from apo-αMT, apo-βMT and Zn7-MT were similar to those from apo-MT. Pb7-MT’ showed better chemical stability than Pb7-MT. The structures of Pb7-MT and Pb7-MT’ were investigated by continuum model method and ONIOM method, respectively. ONIOM method gave better prediction for the absorption spectra of Pb-containing MT. The consistency of computed and experimental spectra suggests that Pb2+binds thiol groups in Pb7-MT by Pb-S3coordination model and in Pb7-MT’by Pb-S3O1coordination model. The ligand O in Pb7-MT’ is from the carboxyl group in the aspartic acid residues.(4) The structure and absorption spectra of As6-MT were investigated by continuous medium model and ONIOM method, respectively. The calculation results indicate that ONIOM method can give better prediction for the absorption spectra of As6-MT. As3+binds thiol groups in As6-MT by As-S3coordination model, which is different from the coordination model of Zn2+in Zn-containing enzyme protein. That will change the structure of the enzyme protein structure. Therefore, after the replacement of Zn2+by As3+, the changed structures of Zn-containing enzyme proteins may induce the disability on their biological functions.