Study On The Interaction Of Heavy Metal Cations Hg～(2+) And Ag～(+) With Spinach Photosystem Ⅱ 33 KD Manganese-stabilizing Protein

The interaction of heavy metal cations Ag+ and Hg2+ with 33 kD manganese stabilizing protein in spinach photosystem II was intensively studied by means of spectroscopy and bioinformatics. Firstly, we analyzed the kinetics, thermodynamics and the conformational changes of the protein induced by binding of metal ions through fluorescence spectra, Ultraviolet differential spectra and circular dichroism spectra. Under different pH values (2.0-9.0), the interaction of metal ions with 33 kD protein was very different as reflected by distinct spectral characteristics. The intrinsic fluorescence of the protein was quenched by Hg2+ in all pH conditions while Ag+ increased the fluorescence intensity at pH 2.0, suggesting different mechanisms for quenching induced by the two metal ions. And the maximum fluorescence emission was slightly influenced by binding of metal ions, which is indicative of little if any effect on the microenvironment of aromatic amino acid residues of metal ions. Distinguished ligand-to-metal charge transition (LMCT) bands were observed through UV differential spectra. The LMCT bands in Hg2+ systems exhibited the same shape and changing pattern under different pH conditions, and the absorption values increased as pH decreased. However, the LMCT bands in Ag+ system displayed distinct changing pattern as the function of pH; and when pH is 2.0, negative signals emerged, which is rarely reported before. The results of LMCTbands indicated that there exist many binding sites in 33 kD protein for Ag+ and Hg2+. Stern-Volmer analysis of the quenching data at pH 7.0 revealed that the quenching induced by Ag+ or Hg2+ was mainly due to static quenching or non-radioactive energy transfer. Further study showed that the number of Ag+ or Hg2+ binding sites for the tryptophan fluorescence quenching was only one, respectively. And the Hg center was very likely to coordinate with the thiol groups of the two cysteine residues; while in addition to cysteine residues, Ag+ also coordinated with tryptophan residue. Thermodynamic analysis showed that the Gibss' free energy (AG) was negative, suggesting the spontaneity of the reaction. Fluorescence emission spectra of 8-Anilino-1-naphthalene-sulfonic acid added to 33 kD protein showed that the protein had an acid expansion under low pH values. CD spectra indicated the protein was dominated by β-sheet (about 41%) and only 4% α-helix. And incubation with Ag+ or Hg2+ resulted in slight conformational changes in secondary structure, exhibiting a little increase in unordered coil. Bioinformatic method was also introcuced to reveal the metal binding sites. According to the coordinate property of both metal ions, it can be speculated that the most possible binding sites for Ag+ and Hg2+ were the thiol groups in cysteine residues, the deprotonated carboxylic oxygens in glutamic and aspartic residues, the amide nitrogens and carboxylic oxygens in glutamine and asparagine residues, and a few deprotonated peptide oxygens. As for Ag+, in addition, side chains of aromatic amino acids, especially the tryptophan residue, were also potential binding sites.