利用重組鋅-乙炔苯酸紫質與螢光團共價修飾之肌紅蛋白突變株作為雙核的光催化系統進行其光能轉化學能之研究

Abstract

隨著化石燃料快速的被消耗，發展替代能源成為全球之共同趨勢，由於太陽能具有源源不絕的特性，因此如何利用太陽能成為替代能源中最受矚目的研究之一。在實驗室先前的研究當中，模擬自然界光合作用而架構出仿生之半人工複合的蛋白質系統，利用生物性材料-將野生型及突變型之脫輔基肌紅蛋白 (apo-myoglobin) 重組入輔基-鋅原紫質 (ZnPP) 與鋅-乙炔苯酸紫質 (ZnPE1)，並應用於光能轉化學能的研究，結果顯示以ZnPE1-MbV68L 之效率為最佳。由於蛋白質在光催化作用的過程中受光能及熱能的影響導致二級結構鬆散致使活性區域內的 ZnPE1 被釋放到緩衝溶液中造成紫質凝集而失去活性，因此如何提升此系統之光能轉化學能的效率成為值得探討的問題。 藉由將不同螢光團 (即I14, P28, Eosin, Texas Red) 共價修飾到不同突變型之重組的鋅-乙炔苯酸紫質 (ZnPE1) 肌紅蛋白，利用螢光團的輔助使整個系統之吸光頻譜被拓展，預期可增進光能轉化學能之效率。利用蛋白質膠體電泳螢光圖 (Fluorescence image of SDS-PAGE)、基質輔助雷射解析電離飛行時間質譜 (MALDI-TOF)、紫外光-可見光光譜 (UV-Vis)、螢光光譜 (Fluorescence) 及圓二色光譜儀 (CD) 分析其生物物理特性以確定螢光團及輔基團皆成功修飾及重組至蛋白質活性區域，利用循環伏安法及微差脈衝伏安法求出螢光團及輔基團之HOMO/LUMO電位。進一步，在光能-化學能轉換的研究，利用不同螢光修飾之重組的鋅-乙炔苯酸紫質肌紅蛋白 (Fluorophore-ZnPE1Mb) 作為光感酵素、菸草醯胺腺嘌呤二核苷酸磷酸鹽 (NADP+) 為受質，三乙醇胺 (TEOA) 為電子提供者之下進行照光反應，可觀察到能量轉換的現象。 比較與分析結果後發現，在螢光團及輔基團皆被適當波長的光激發時較被不適當波長的光激發時增進約30% 之效率，此外，此拓展吸光頻譜之雙核的光催化系統亦可作為新一代光能轉換系統的設計概念。

Currently, the world’s supply of fossil fuels is being consumed at an alarming rate, and, because sunlight is plentiful, solar power has become one of the most popular areas in the development of alternative, renewable energy sources. In a previous study, an artificial, protein-based, photo-chemical, energy-conversion system, which mimicked a natural photosystem, was constructed by using ZnPP-Myoglobin/ZnPE1-Myoglobin as a photocatalyst, and the results showed that ZnPE1-MbV68L had the best catalytic velocity. During photoirradiation, the relaxation of the secondary structure caused by light and thermal energy (due to temperature increases) causes the decomposition of photoenzymes, releasing porphyrin from the heme pocket and causing the aggregation effect in the buffer solution. Consequently, the function of the photoenzymes will be lost, creating the need to improve photocatalytic velocity during the limited lifetime of the photoenzymes. The strategy of using various fluorophores (i.e., I14, P28, Eosin and Texas Red) covalently conjugated to the reconstituted ZnPE1-Mb mutants because of the absorption of fluorophores can assist in extending the absorbance region, and it can be assumed that the construction of the Core Duo photocatalytic complex will improve the catalytic velocity of the conversion of light energy to chemical energy. In addition, fluorescence images of SDS-PAGE, MALDI-TOF, and UV-Vis/fluorescence spectra and circular dichroism (CD) have been used to characterize the biophysical and optic properties of the fluorophore-ZnPE1Mb mutants, which confirmed that the fluorophores and prosthetic groups have been conjugated and reconstituted successfully in the correct location in heme pocket of Mb. In addition, the redox potential of fluorophore-ZnPE1Mb mutants was confirmed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques to estimate the energy level between the fluorophores and ZnPE1, and the results showed that the Core Duo photocatalytic system functions reasonably. Next, we performed the conversion of light energy to chemical energy of NADP+ reduction utilizing different fluorophore-ZnPE1Mb mutants as the photoenzyme and photoirradiated at 352/419, 419/419, and 419/580 nm wavelengths, respectively. The energy conversion can be observed in the artificial photocatalytic system. To summarize, based on the results of photocatalytic velocity, the velocity of the NADP+ reduction using suitable wavelength light for excitation of every fluorophore-ZnPE1Mbmut is approximately 30% greater than that of the system using lights of non-suitable wavelengths. Moreover, the concept of this artificial Core Duo photocatalytic system that can absorb light in the widest possible wavelength region will be realized as a potential bio-photosentizer and applied as a more efficient photocatalytic system for the development of renewable energy due, in part, to the very important information developed in this study.