The Prepartion And Studies On Metal Porphyrin Recombination Neuroglobin

Design, synthesis, property and function investigation of artificialmetalloproteins/metalloenzymes have become one of the most important topics in the fieldof bioinorganic chemistry. In recent years, some artificial metalloproteins/metalloenzymesbased on the peptides of hemoproteins such as hemoglobin (Hb), myoglobin (Mb) andhorseradish peroxidase (HPR) have been reported. The recently discovered neuroglobin(NGB) is a novel six-coordinated hemoprotein. NGB has the typical globin fold despite thevery limited (≈20–25%) similarity in amino acid sequence when compared with vertebrateMb and Hb. So far, artificial metalloprotein/metalloenzyme based on the polypeptide chainof NGB has been rarely reported. In this thesis, we have prepared wild and several mutantpolypeptide chains of NGB by using genetic engineering, site-directed mutagenesis ofprotein and biochemical methods. Then, manganese-substituted and cobalt-substitutedrecombinant metalloproteins/metalloenzymes were obtained by incorporation ofmetalloporphyrin into these polypeptide chain scaffolds. Further, we have investigatedtheir nitrite reductase activities, electrochemical behaviors and defolding/refoldingproperties. The main methods and results are as the followings:1. The preparation of manganese-and cobalt-recombinant NGB and its mutant proteins(H64Q-NGB，H64V-NGB). The pET3a plasmid with the gene of NGB and its mutantproteins (H64Q，H64V) were transformed to E. Coli BL21(DE3) plys cells and expressedin TB culture medium. Lysates were treated with ammonium sulfate precipitation,DEAE-Sepharose anion exchange column, Hiload16/60，superdex75size exclusionchromatography and a Hiprep16/10QFF anion exchange column, respectively, the targetred soluble proteins were purified and obtained.Apo-NGB, apo-H64Q-NGB and H64V-NGB polypeptide chains were prepared by themethod of acid-acetone. Manganese-porphyrin and cobalt-porphyrin recombinantmetalloproteins/metalloenzymes were obtained by incorporation of metalloporphyrin into these polypeptide chain scaffolds.2. The nitrite reductase activity of Mn-NGB and Co-NGB. The interactions betweenMn(III)-NGB and NO2-were weak, the coordination environment around the metalporphyrin changed slightly. In anaerobic conditions, there were strong interactions betweenMn(II)-NGB and NO2, then Mn(II)-NGB(NO) was formed. We studied several factorseffecting the reaction activity such as the concentrations of Mn(II)-NGB, NO2and pH.The result indicating that in the pH range of6~8, lower pH favored the reaction activity.With increased concentrations of Mn(II)-NGB, NO2, the reaction activity increased.Thenitrite reductase activity of Co-NGB indicated that the interactions between Co-NGB andNO2-were weak. the coordination environment around the metal porphyrin changedslightly. It was harder to have Co(III)-NGB reduced than Mn-NGB. The interactionsbetween Co(II)-NGB and NaNO2, were much slower and weaker than that of Mn(II)-NGB.3. The nitrite reductase activity of Manganese reconstituted H64Q and H64V, Cobaltreconsitued H64Q. The interactions between Mn(III)-H64Q and Mn(III)-H64V with NO2were weak, respectively. n anaerobic conditions, the coordination environment around themetal porphyrin changed slightly. In anaerobic conditions, the reactions betweenMn(II)-H64Q and NO2were much faster than that of Mn(II)-NGB.The reaction proceededas followings: Mn(II)-H64Q+NO2Mn(II)-H64Q(ONO) Mn(II)-H64Q(NO). Thatthe distal His was changed to Val affected the nitrite reductase activity greatly. The timethat reached equilibrium was wt-NGB (10min), Mn(II)–NGB (28min), Mn(II)–H64Q (25min), Mn(II)–H64V (7.5min)。When the distal His was changed to aliphatic amino acidesthe six coordinated state changed to five coordinate state, which increased the nitritereductase activity. It was hard for distal ligand to coordinated with the metal porphrinwhich facilite the interactions between metal porphrin and NO2. The studies indicated thatthe concentrations of Mn(II)–H64Q，Mn(II)–H64Vå'ŒNO2–had little impact on thereaction rates. In the pH range of6-8, the reaction rates increased with lower pH. Theinteractions between Co(II)–H64Qand NO2–were weak.Co(III)–H64Q was easier to bereduced compared with that of Co(III)–NGB. The reaction rate of Co(II)–H64Q with NO2–was larger than that of Mn(II)–H64Q and Mn(II)–H64V, implying that the metal porphrin affect the nitrite reaction activity greatly and that might be account for the electronic spinstate of d.4. The alcohol and methanol induced unfolding and refolding of Co-NGB. In theunfolding procedure, UV-vis absorption spectroscopy indicated that the protein chainsunfolded gradually as the concentrations of alcohol and methanol increased. Thatabsorption at Soret and280nm increased indicated the dissociation of metal porphyrinfrom the protein chains. The fluorescence spectrum of Co-NGB indicated that thefluorescence intensity at348nm increased gradually as the protein chains unfolded and themetal porphrin dissociated from the protein chain. The fluorescence quenching vanished asthe ocurence of fluorescence resonance energy transfer between metal porphrin and proteinchain. The CD indicated that alcohol could induce more content α–helix. In the refoldingprocedure, as alcohol concentrations decreased, there was a little red shift at280nm, andthe Sore absorption decreased. The fluorescence intensity decresed as the protein chainfolded. CD indicated that the negative peaks at208nm and222nm gradually formed. Theprotein gradually got back to normal state. And Co-NGB exhibited the activityofperoxidase.5. The electrochemical behaviors of Co-NGB. we studied the cyclic voltammograms ofCo-NGB.The electron electrode was prepared by3-mercaptopropionic acid and L-cysteine,the electronic response of Co-NGB was obtained. With scan rates increased, there were noapparent changes at the redo potential, indicating a fast transport of electron. As thevoltammetric response decreased in the pH range of6~9, it revealed pH had a greatinfluence on the protein’s electronic response. The electrostatic forcing of Co-NGB willchange if associated with different cationic ion and anionic and the electronic propertieschanged.