Rare Earth And Its Complexes With Microperoxidase Interaction Mechanism

In recent years, the rare earth microfertilizer has been increasingly applied to the agriculture in China and other countries because it can increase the yield and the quality of various agricultural productions. It has been found from the study on the biological machanism that the rare earth ions used at the reasonable concentration can improve the adversity resistance, promote the transformation of the light energy during photosynthesis, stimulate the growth of cells and enhance the biological activities of some enzymes, such as catalase, peroxidase, superoxide dismutase, etc. However, the interaction mechanism is still unclear and the related study is less because these enzemy possess high chemical activities, complex structures and flexible conformations so that the effect of rare earth ions on physiological and biochemical behaviors of biomacromolecules is difficult to be investigated at the molecular levels. Therefore, in this thesis, MP-8 and MP-11, whose active center and bioactivity are similar to POD, was used as the model of POD and Eu³⁺ was used as the typical rare earth ion to investigate the interaction mechanism of MP-11, MP-8 and Hemin with Eu³⁺ using the electrochemical method, the UV-vis absorption spectroscopy and electrospray ionization mass spectrometry. Moreover, the interaction between Eu³⁺ and lanthanide complexes with VB₆- Gly Schiff Base was also studied. All above studies would lay a strong base on the investigation of the interaction mechanism between the rare earth ions and the biomacromolecules. The main results obtained are as follows:1. The interaction mechanism of Eu³⁺ with MP-11(1) When Eu³⁺ is added into the MP-11 solution, Eu³⁺ is prior to coordinate with the carbonyl oxygen of the metacetonic acid group in the heme group of the MP-11 molecule, forming the complex of Eu³⁺ and MP-11 because the carbonyl oxygens of the metacetonic acid group have large exposed extent and the negative charge. On the other hand, the rare earth ions are prone to coordinate with oxygen. The coordination of Eu³⁺ and the two carbonyl oxygens leads to the increase in the non-planarity of the porphyrin group in the heme group, the decrease in the energy and the probability for theπ-π^* transition because the the metacetonic acid group is close to the the porphyrin group in the heme group. Eu³⁺ interacts hardly with the carbonyl oxygen of the amido groups in the peptide chain of the MP-11 molecule because the exposed extent of the carbonyl oxygen in the peptide chain is small due to the formation of the hydrogen bonds in the peptide chain and carbonyl group of peptide chain is barried in hydrophobe group of peptide.(2) The studies of the electrospray ionization mass spectrometry show that when Eu³⁺ is added into the MP-11 solution the molar ratio of Eu³⁺ and MP-11 in the complex formed is 1. No new complexes are formed when the molar ratio of Eu³⁺ and MP-11 is increased, indicating that one Eu³⁺ ion can only coordinate with a MP-11 molecule, forming the complex with two positive charges. The electrochemical experiments also prove this result, i.e. the redox peaks of Eu³⁺ almost disappear and the only the redox peaks of MP-11 are observed when the molar ratio of Eu³⁺ and MP-11 is less than 1 because most of Eu³⁺ ions coordinate with MP-11. If the molar ratio of Eu³⁺ and MP-11 is larger than 1, a pair of the redox peaks of Eu³⁺ appears again in the cyclic voltammogram except the redox peaks of MP-11, indicating that the surplus Eu³⁺ ions do not coordinate with MP-11. Thus, the molar ratio of Eu³⁺ and MP-11 in the complex formed is 1.(3) When Eu³⁺ forms the complex with MP-11△Ep of the complex in the cyclic voltammograms is smaller and the peak currents are larger than that of MP-11, indicating that Eu³⁺ can promote the electrochemical reaction of MP-11. The reason for this is that the interaction between Eu³⁺ and Mp-11 can increase the nonplanarity of the heme group of MP-11 and the exposure degree of Fe(III) in the heme group due to that Eu³⁺ can coordinate strongly with the carbonyl oxygen of the metacetonic acid group in the heme group of the MP-11 molecule. On the other hand, the electrocatalytic activity of the complex for the reduction of oxygen is higher than that of MP-11, illustrating that Eu³⁺ can increase the biologic activity of MP-11.2. Effects of peptide structure of microperoxidase on the interaction between Eu³⁺ and MicroperoxidaseIn this section, the interaction of Eu³⁺ with Hemin Mp-8 Mp-11 and Cyt-c was studied in order to investigate effects of peptide structure on the interaction between Eu³⁺ and microperoxidases. It can lay a solide foundation on further investigation of the interaction mechanism between rare earth and microperoxidase.(1) The UV-vis spectroscopic studies illustrate that the peak positions andabsorbances of all the Soret bands of Hemin Mp-8 Mp-11 and Cyt-c are red-shifted and dcreaseed, respectively after they interacte with Eu³⁺. Among them, the change of Hemin in the peak position and absorbance of the Soret bands is the largest. The reason for this is that the porphyrin ring of Hemin has no peptides to be connected, so that its negtive charged carbonyl oxygens of the metacetonic acid group are easy to be coordinated with Eu³⁺. While, the peptide of cyt c is long and complicated so that the heme group is buried in the peptide of cyt c and Eu³⁺ is difficult to coordinate with the carbonyl oxygen of the metacetonic acid group in the heme group. Thus, the change of Cyt c in the peak position and absorbance of the Soret bands is the smallest. Peptides length of MP-8 and MP-11 is different, which leading their different aggregrative structures. MP-11 exhibits preferentially an irregular alignment (so-called head-to-tail) of the heme planes and each heme plane is not parallel, while MP-8 molecules are aggregated in a more regular stacked conformation, leading the nonplanarity of Mp-11 is larger than Mp-8. Thus, reacted with Eu³⁺, the heme plane of Mp-11 distorts much more than Mp-8's. The Soret bands of Mp-8 and Mp-11 are located at 396 and 400nm respectively, reflecting their different plane characters. Therefore, the red shift of the Soret band decreases in the order of Hemin > MP-11 > MP-8 > Cyt-c, while the decrease in the absorbance of the Soret band is in the order of Hemin > MP-8 > MP-11 > Cyt-c.(2) The electrochemical studies show that Eu³⁺ can coordinate with Hemin, MP-8 and MP-11, respectively, forming the complex with the molar ratio of 1. Although the coordination between Eu³⁺ and Hemin is the strongest, the redox peaks of the complex of Eu³⁺ and Hemin can not be observed in the cyclic voltammogram of the complex, illustrating that Eu³⁺ would inhibit the electrochemical reaction of Hemin. For Mp-8 and Mp-11, the electrochemical reversibility in the present of Eu³⁺ is better than that withou Eu³⁺, indicating Eu³⁺ can promote their electrochemical reaction. Because the aggregation of MP-11 is more incompact than that of MP-8, the increase in the nonplanarity for MP-11 caused by the coordination of Eu³⁺ is larger than that for MP-8. Then, the promote effect of Eu³⁺ for the electrochemical reaction of MP-11 is larger than that of MP-8. Eu³⁺ cannot coordinate with the heme group in Cyt c because the heme group is buried in the peptide of cyt c. Thus, Eu³⁺ cannot affect the electrochemical reaction of Cyt c.3. Interaction of MP-11 and La-VB₆-Gly complexIn this section, MP-11 was selected as the POD model compound to investigate systemly the interaction between MP-11 and La-VB6-Gly (LaLCl₂) complex using the UV-vis absorption spectroscopy, CD spectroscopy and cyclic voltammetry.(1) The UV-vis absorption spectroscopic results indicate that the peak position of the Soret band of MP-11 is not changed and its absorbance is increased when the molar ratio of LaLCl-2 and MP-11 is lower than 5. When the molar ratio is larger than 10 the peak position of the Soret band is red-shifted and its absorbance is decreased. It may demonstrate that there are two interaction modes between MP-11 and LaLCl₂, i.e. the promote mode for low molar ratio and the restraint mode for the high molar ratio. The coordination between MP-11 and LaLCl₂ leads to the increase in the nonplanarity and the explosure of hemin as well as the decrease in the probability and the energy ofπ-π^* electron transition.(2) The CD spectroscopic results show that the addtion of LaLCl₂ would affect the second structure of the peptides in MP-11, leading the red shift of the Soret band and the decrease of the molar ellipticity. With the increase in the complex concentration, at first, the ordered structure of MP-11 is increased and then decreased, indicating the breakage of the ordered second structure and the further increase in the nonplanarity of the heme group in MP-11 with increasing the complex concentration. (3) The electrochemical experiments find that the electrochemical reaction of the complex of MP-11 and LaLCl₂ is a quasi-reversible and diffusion-controlled. LaLCl₂ can promote the electrochemical reaction of MP-11 at the molar ratio of 3. However, this promote action would be decreased with the further increase in the molar ratio. The above results reflect that their maybe two kinds of the interaction mode between MP-11 and LaLCl₂In conclusion, the La-VB₆-Gly complex can coordinate with MP-11 directly, leading to the change in the second structure of MP-11 peptide, the increase in the nonplanarity of the heme group of MP-11 and promoting the electrochemical reaction of MP-11.