人工重組鋅紫質-肌紅蛋白系統應用於光催化能量轉換之研究

Abstract

現今，地球的化石燃料 (煤炭、石油和天然氣等) 正以每年2000萬年儲存量的驚人速度被使用著；以這樣的速度，地球上的化石燃料將很快的被消耗殆盡。許多科學家們開始致力於替代能源的開發與研究，其中以太陽能來說，其照射地球一小時的能量 (4.3 x 1020 J/hour) 就相當於地球人類一年的使用量 (4.1 x 1020 J) ，再加上其生產過程無環境污染，又不會消耗其他地球資源或導致地球溫室效應的優點，這樣的「綠色能源」被視為現今替代能源的開發重點。然而以自然界來說，能最有效率直接將光子轉換成電子並進而轉換成化學能的系統就是綠色植物及光合作用菌，而這樣有效率的系統基本上是由可吸收光的輔基及蛋白質催化的電子轉移機制所組成的。於是我們基於環保能源及仿生的概念去架構一個半人工複合的蛋白質系統，我們利用生物性材料-脫輔基肌紅蛋白 (apo-myoglobin) 去重組具有不同結構及吸光特性的輔基-鋅原紫質 (ZnPP) 與鋅-乙炔苯酸紫質 (ZnPE1) ，並將這些重組的鋅原紫質與鋅-乙炔苯酸紫質肌紅蛋白 (ZnPP-Mb and ZnPE1-Mb) 當做模板蛋白 (model protein) 去應用於光能轉化學能、光能轉電能與光能產氫方面的研究。 首先，我們已成功的將鋅原紫質 (ZnPP) 與鋅-乙炔苯酸紫質(ZnPE1) 重組進脫輔基肌紅蛋白 (apo-myoglobin) 並利用紫外光-可見光光譜 (UV-Vis) 、螢光光譜 (Fluorescence) 、圓二色光譜儀 (CD) 、時間-解析螢光光譜 (TCSPC) 去分析其物理與光學特性，及利用循環伏安法 (CV) 及微差脈衝伏安法 (DPV) 去測得其氧化還原電位。在時間瞬態光譜研究方面，我們研究了ZnPP/ZnPE1在四氫呋喃 (THF) 、磷酸鉀緩衝溶液 (KPi) 及包覆進肌紅蛋白 (myoglobin) 等不同環境下的電子受激發後緩解的現象。證實當鋅原紫質 (ZnPP) 與鋅-乙炔苯酸紫質( ZnPE1) 被包覆到肌紅蛋白 (myoglobin) 時，能延長光敏化劑受光激發的生命期並在水相環境中能有效避免聚集現象的發生。 進一步，在光能-化學能轉換的研究方面，利用所架構的半人工複合蛋白質系統結合可氧化還原的受質以研究光激發電子傳遞的機制，當重組的鋅原紫質與鋅-乙炔苯酸紫質肌紅蛋白 (ZnPP-Mb and ZnPE1-Mb) 結合氧化態菸草醯胺腺嘌呤二核苷酸磷酸鹽 (NADP+) 與還原態菸鹼醯胺腺嘌呤雙核苷酸 (NADH) 受質，在有電子提供者及缺乏電子提供者存在下照光反應，可觀察到電子轉移及能量轉換的現象。證實此半人工複合蛋白質系統具有發展研究光化學電池的潛力。 在光能轉換電能的部分，已成功的將鋅紫質肌紅蛋白 (ZnPP-Mb) 修飾吸光團-伊紅 (Eosin) ，並比較其與野生型肌紅蛋白、鋅紫質肌紅蛋白之光電轉換效率。結果發現在結合二氧化鈦陽極 (TiO2 anode) 、水相電解液及白金陰極 (Pt cathode) 的染敏太陽能電池 (DSSC) 系統照光下，可觀察到光電流的產生。以伊紅修飾之重組鋅紫質肌紅蛋白之光電轉換效率高於野生型肌紅蛋白及鋅紫質肌紅蛋白。 光能產氫方面的研究方面，利用鋅原紫質與鋅乙炔苯酸紫質肌紅蛋白 (ZnPP-Mb and ZnPE1-Mb) 當作光敏劑結合電子提供者 (electron donor) 、電子繼電者 (electron relayer) 及白金催化劑 (Pt catalyst) 在受光激發下可觀察到氫氣的產生。另外在氫氣催化劑的研究方面，近年來利用結晶學的技術，科學家已成功地解出去磺弧菌 (Desulfovibrio desulfurican) 以及巴斯德氏梭狀芽胞桿菌 (Clostridium pasteurianum) 中鐵-鐵產氫酶 ([FeFe]-Hydrogenase) 的X-ray晶體結構。而這類酵素活化中心的氫簇分子 (H-cluster) 就是使其具有高效能產生氫氣的催化中心。我們進一步模擬並合成出鐵-鐵產氫酶活化中心-氫簇分子(H-cluster)的化學結構-[(μ-DT)Fe2(CO)6] (DT: dithiolate) ，並在其雙硫架橋上取代成不同的官能基及加入不同的磷衍生物並利用有機相和水相兩種不同的反應系統來探討這些仿生酵素活化中心催化氫氣產生的效率以及機制。在之後預期能合併鋅原紫質與鋅-乙炔苯酸紫質肌紅蛋白 (ZnPP-Mb and ZnPE1-Mb) 與仿生合成之硫鐵簇分子 (iron-sulfur cluster) 去架構一個新的光催化仿生產氫系統。

Today, world storage of fossil fuels such as coal, oil and natural gas is consumed with the alarming rate, 20 million years storage capacity per year. There is no doubt that fossil fuels will soon be depleted. As a result, many scientists have started to focus on development and research of alternative energy. Sunlight is not only the most obvious but also most predominant renewable alternative energy source on Earth. When solar energy strikes the Earth's surface for one hour (4.3 x 1020 J/hour), it is more than all human-related energy consumption on the planet for one year (4.1 x 1020 J). In addition, it does not consume any other energy source and does not cause pollution or the “green house effect” in production. This kind of “green energy” is a considered key to develop alternative energy. In nature, photosynthesis, which is composed by chromospheres and redox protein with electron transfer activity, is the most universal, effective and important photo-chemical energy conversion system. This process is based on conversion of solar energy into chemical energy by a series of intermolecular energy and electron transfer reactions. Based on the “green energy” and biomimetic concept, an artificial metalloporphyrin-myoglobin complex has been constructed. Zinc protoporphyrin (ZnPP) and 5-(4-carboxy-phenylethynyl)-10,20-biphenylporphinato zinc (II) (ZnPE1) have been reconstituted into apo-myoglobin (apo-Mb) and have been applied as model proteins to light-to-chemical energy, light-to-electric energy, and light-to-hydrogen conversion studies. In addition, UV-Vis, fluorescence, circular dichroism (CD), and time-correlated single-photon counting (TCSPC) spectra analysis have been used to characterize the biophysical and optic properties of these reconstituted ZnPP-Mb/ZnPE1-Mb. The redox potential of ZnPP-Mb/ZnPE1-Mb was further confirmed by cyclic voltammetry (CV) and differential pulse voltammetry techniques (DPV). The fluorescence transients exhibited a biphasic decay feature with the signal approaching an asymptotic offset: at λem = 630 nm, two time constants of τ1 = 0.5 ns and τ2 = 2.2 ns for ZnPP-Mb and τ1 = 0.6 ns and τ2 = 2.5 ns for ZnPE1-Mb, respectively. In studies of light-to-chemical energy conversion, an artificial protein-based photo-chemical energy conversion system which mimics photosystem I has been constructed using ZnPP-Mb/ZnPE1-Mb as a photosensitizer, nicotinamide adenine dinucleotide phosphate (NADP+) as an electron acceptor, and triethanolamine (TEOA) as a sacrificial electron donor. Moreover, this artificial system can proceed the reverse oxidation reaction by providing nicotinamide adenine dinucleotide (NADH) as electron donor and in absence of TEOA. Apo-Mb is a key factor to keep monomeric ZnPP/ZnPE1 in buffer solution, prevent aggregated quenching, and improve the efficiency in photo-induced redox reaction. In studies of light-to-electric energy conversion, Mb, ZnPP-Mb, and Eo-ZnPP-Mb were used as photosensitizers to functionalize TiO2 nanocrystalline films for biosensitized solar-cell (BSSC) applications. For the Mb-sensitized solar cell, poor cell performance is due to a reduction Fe(III) → Fe(II) that produces a photocurrent density of a device that is smaller than its unsensitized counterpart. The efficiencies of power conversion and photocurrent for both ZnPP-Mb and Eo-ZnPP-Mb–sensitized solar cell are enhanced about ten times due to superior charge separation between TiO2 and the protein, and due to smaller current leakage between TiO2 and the electrolyte. The cell performances of the BSSC devices are discussed in terms of an equivalent-circuit model. In studies of light-to-hydrogen conversion, a photocatalytic hydrogen generation system has been constructed using ZnPP-Mb (or ZnPE1-Mb) as photosensitizers to combine the electron donor, electron relayer, and catalyst. Catalysts that mimic hydrogenases have also been investigated. The crystal structures of one kind of hydrogenases, [FeFe]-hydrogenases from Desulfovibrio desulfuricans and Clostridium pasteurianum, have been elucidated by X-ray crystallography. The organometallic H-cluster unit of its active site is involved in water splitting and provides a very high rate of hydrogen generation. We try to mimic the structure of the H-cluster, [(μ-DT)Fe2(CO)6] (DT: dithiolate) in [FeFe] hydrogenases by replacement of bridging dithiolate ligands and modification of various phosphine ligands to study their catalytic activity for hydrogen production. Furthermore, the efficiency of photocatalytic hydrogen generation using these compounds as catalysts are investigated in aqueous and organic phase systems. It is expected to conjugate ZnPP-Mb (or ZnPE1-Mb) with artificial iron-sulfur clusters in a homogeneous catalytic system to further investigate light-driven hydrogen production activity.