Study On The Influence Of Surface Porperties On Direct Electron Transfer And Adsorption Kinetics Of Cytochrome C

Electron transfer?ET?in biological system is one of the most basic processes in biological energy transduction and conversion.In organism,the electron transfer reaction of many redox proteins occurs at the charged cell biomembrane surface,so their electron transport is inevitably affected by the membrane potential.This effect can be studied by the electrochemical interfaces.This study will help to understand the protein-interface interaction,and provide theoretical and experimental bases for understanding the electron transfer mechanism of proteins in life,and also for developing new bioelectrocatalytic system and electrochemical biosensors.The immobilized amount,conformation and orientation of adsorbed proteins show great influence on the direct electron transfer capability between adsorbed redox protein and the underlying electrode.However,there exists controversial conclusion about the dependence of direct electron transfer activity of protein on its heme plane orientation.In view of the existing problems on how the direct electron transfer capability and electrocatalytic activity of hemeprotein depends on its heme plane orientation,this thesis carried out the following four aspects:1.Direct electrochemistry of cytochrome c on a graphene/poly?3,4-ethylenedioxythiophene?nanocomposite modified electrodeIn this work,the direct electrochemistry of cytochrome c?cyt c?embedded in a novel support matrix of graphene/poly?3,4-ethylenedioxythiophene??G-PEDOT?nanocomposite on a glassy carbon electrode was studied.The prepared G-PEDOT nanocomposite is of good biocompatibility,enhanced electric conductivity and large specific surface area.The immobilized cyt c in G-PEDOT matrix displays excellent direct electrochemistry and retains its biocatalytic activity toward the reduction of hydrogen peroxide.The electrochemical signal shows a linear response to H2O2 in the concentration range from 5.0×10-7 to 4.0×10-4 M with a detection limit of 2.49×10-7 M.The results demonstrate that the G-PEDOT nanocomposite offers a biocompatible material for the construction of biofuel cells,bioelectronics and biosensors.2.Direct electrochemical study of cytochrome c at SAMs/Au electrodes with different wettability-heme plane orientation dependent electron transferThe effects of interfacial wettability on the structure-funcion relationship of assembled protein were explored.Five types of SAMs modified gold electrode surfaces with different wettability were fabricated by varying the stoichiometric ratio of the hydrophilic thiol molecule to hydrophobic thiol molecule.We studied the adsorbed amount,conformation,molecule orientation and the direct electron transfer activity of cyt c on these electrode surfaces.The factors such as the immobilized amount,conformation and orientation,distance between the active center of protein and the electrode that cause the different electrochemical activity of the assembled cyt c on these surfaces with different wettability were analyzed.The results indicate that the direct electron transfer is dependent on the orientation of heme plane in assembled cyt c.Orientation with heme plane in cyt c parallel to gold electrode surface favors the direct electron transfer,while vertical orientation of heme plane makes the direct electron transfer difficult.A preferable electron transport pathway for cyt c is through the axial ligands(His^-18)of the heme center rather than the porphyrin ring.3.Stuuy on eiecirocnemicai activity of nemin uepenuent on neme prane interfacial orientationIn view of the existing problems on how the direct electron transfer capability and electrocatalytic activity of hemeprotein depends on its heme plane orientation,we propose a strategy to control directly the orientation of hemin via experimental models,Hemin-His model and Hemin-MHN model,and then study the structure-function relationship of the assembled hemin.In the Hemin-His model,the heme plane orients parallel to the modified electrode surface via coordination bond between the pyridinic nitrogen in His on the electrode surface and the iron center of hemin.While in the Hemin-MHN model,covalently bound hemin prefers to orient with its heme plane relatively vertical to the modified electrode surface.Electrochemical results indicate that the direct electron efficiency of hemin is highly dependent on the heme plane orientation.In addition,the electrocatalytic reduction mechanism of H2O2 by hemin in the Hemin-His model was also explored by spectroelectrochemical technique.This work further supports the concept of heme plane orientation dependent direct electrochemical behaviors of redox protein in our previous work.4.Dependence of the direct electron transfer activity and adsorption kinetics of cytochrome c on interfacial charge propertiesWith the advantages of in-situ analysis and high surface sensitivity,surface enhanced infrared absorption spectroscopy in attenuated total reflection mode?ATR-SEIRAS?combined with electrochemical methods has been employed to examine the interfacial direct electron transfer activity and adsorption kinetics of cyt c.This work presents data on cyt c adsorption onto negatively charged mercaptohexanoic acid?MHA?and positively charged 6-amino^-1-hexanethiol?MHN?self-assembled monolayers?SAMs?on gold nanofilm surfaces.The adsorbed cyt c displays the higher apparent electron transfer rate constant(33.5±2.4 s^-1)and apparent binding rate constant(73.1±5.2 M^-1·s^-1)at the MHA SAMs surface than those on the MHN SAMs surface.The results demonstrate that the surface charge density determines the protein adsorption kinetics,while the surface charge character determines the conformation and orientation of proteins assembled which in turn affects the direct electron transfer activity.