Assembly And Properties Of Catalase Ordered Composite System

Hydrogenases(H₂ases)can catalyze reversible oxidation of molecular hydrogen into protons and electrons.They are widely distributed among microorganisms,with the capacity either to use hydrogen as an energy source or to dissipate excess reducing equivalents in the form of molecular hydrogen.Under the circumstance of global environmental and energy crisis,it is now well worth using the photosynthetic proteins,photosystemâ… (PSâ… ),photosystemâ…¡(PSâ…¡)as well as H₂ase to mimic the biological route to split water and produce clean energy of hydrogen gas.To well understand the hydrogenase catalytic hydrogen evolution process and prepare artificial systems for the energy transfer,proteins are usually immobilized on substrate surfaces.It has been considered that the organized ultrathin films of H₂ase play an important role in the preparation of fuel cells,biohydrogen and nano-device. The purpose of the present work was design and preparation of hydrogenase ultrathin films for the biohydrogen evolution.The well-developed molecular assembly methods of Langmuir-Blodgett(LB),self-assembly and layer-by-layer were used for the assembly of H₂ase organized ultrathin films on quartz,mica and ITO substrate surfaces.These films were characterized,with detailed studies on the optical and electrochemical properties of the proteins and related compounds.The details are summarized as follows.To prepare carbon nanotubes(CNYs)-H₂ase cojugates,the commercial available solid CNTs were first oxidized and dissolved in water,which was then mixed with H₂ases in a given concentration.The oxidized CNTs were characterized using Raman, infrared and adsorption spectra,the features of which indicated that the CNTs obtained were covered by plenty of COOH groups.The CNTs-H₂ase conjugates were prepared by mixing H₂ase with CNTs-saturated aqueous solution in the ratio of 1;1 (v/v).These conjugates were spread on to Tris-HCl subphase surface to form stable Langmuir monolayers of hydrogenase.With the use of LB technique,the CNTs-H₂ase monolayers were transferred onto substrate surfaces,which were characterized by FTIR and absorption spectra,as well as transmission electron microscopy.The ITO electrodes modified with the LB films of CNTs-H₂ase conjugates revealed well reversible redox waves corresponding to the redox reaction of[4Fe-4S]^2+/1+clusters in the H₂ase.The current intensity was enhanced after co-assembly with CNTs.Because of the different diameters of the CNTs used,this current intensity was proportional to the scan rate(ν)for the electrodes modified with the LB films of pure H₂ase and single-walled CNTs(SWNTs)-H₂ase,but to the root of scan rate(ν^1/2)for those modified with the multi-walled CNTs(MWNTs)-H₂ase LB films.The products of diffusion coefficient and concentration(D^1/2C)increased in the order of pure H₂ase, SWNTs-H₂ase,and MWNTs-H₂ase LB films.The results suggested that the co-existed CNTs could not only support the formation of stable hydrogenase films but also enhance the electron transfer efficiency between the electrode surfaces and the active centers of the hydrogenase.Besides with the use of LB technique,we also used the casting technology to prepare the CNTs-H₂ase conjugates modified glass carbon(GC)electrodes.Here, both the oxidized CNTs and Nation-soluble CNTs were used as the supporting layer to adsorb H₂ase layers on the GC electrode surfaces.It was found that the pure H₂ase formed dot-like domains,while the oxidized CNTs-H₂ase and Nafion-CNTs-H₂ase composites formed wire and large closely packed aggregates,respectively.The reduction potentials for the cluster of[4Fe-4S]^2+/1+of H₂ase were at about-500,-650 and-700 mV(vs Ag/AgCl)for the electrodes modified with pure H₂ase, Nafion-SWNTs-H₂ase and Nation-MWNTs-H₂ase,respectively.Potential step chronocoulemetry measurements indicated a larger charge transfer diffusion coefficient when CNTs were co-assembled,suggesting that CNTs can not only act as a supporting layer to immobilize enzymes,but also act as a highly conductive wire throughout the films.Thirdly,two kinds of light-sensitive,electroactive and enzyme active electrodes modified by zinc porphyrins,hydrogenase,and viologen triad multilayers have been constructed.The first zinc porphyrin or viologen layer was covalently immobilized on the substrate surfaces,with the consecutive layers of hydrogenase and polyviologen or positively chagred porphyrin adsorbed electrostatically.The multilayer assembly was characterized by using X-ray photoelectron and UV-vis absorption spectroscopy. Electrochemical studies for the zinc porphyrin modified electrode showed a couple of redox wave centered at about-0.48 V vs Ag/AgCl,which was attributed to a bielectronic process of porphyrin.When the first layer was viologen derivative(VIO), two couples of redox waves were recorded and centered at about-0.40 and-0.86V, which were attributed to one electron processes of VIO²⁺←→VIO^+·and VIO^+·←→VIO⁰,respectively.The hydrogenase activity was remained in the multilayers.The as-prepared multilayers were a three-component system composed of photosensitizer, catalyst and electron mediator,resulting in potential interests in the studies of light-induced energy/electron transfer in the molecular level as well as in the development of molecular devices for the biohydrogen evolution,hydrogen gas biosensors and biofuel cells.Fourthly,monolayer behaviors and electrochemical properties for the conjugates of CNTs-cytochrome c(Cyt c)were investigated.We also found that both the SWNTs and MWNTs were able to support the formation of the conjugate monolayers.Surface pressure-area isotherms showed an enlargement for the Cyt c occupied area when CNTs-Cyt c conjugates were spread on the subphase surfaces.Area-time isotherms indicated that the MWCNTs-Cyt c monolayer was more stable than the pure Cyt c and SWCNTs-Cyt c monolayers.With the use of Langmuir-Blodgett(LB)method, (CNTs-)Cyt c monolayers were transferred on to electrode surfaces.Electrochemical measurements for the(CNTs-)Cyt c LB film-modified electrodes showed well reversible redox waves of Cyt c.Compared to that of the pure Cyt c LB film,the redox current intensity was increased for the CNTs-Cyt c conjugate LB film modified electrodes.The results suggest that CNTs can not only support the formation of ultrathin Cyt c LB films,but also enhance the electron transfer between electrode and proteins.Finally,we constructed composite films containing hydrogenase(catalyst)and zinc porphyrin(photosensitizer)on the quartz and ITO substrate surface by the LB method. Spectral and morphological features confirmed that the zinc porphyrin was adsorbed surrounding the H₂ase and forming aggregates.Electrochemical studies for the ITO electrodes modified by the composite LB films revealed quasi-reversible redox processes of both hydrogenase and porphyrin.