利用重組鋅-原紫質及鋅-乙炔苯酸紫質之不同肌紅蛋白突變株進行其光能轉化學能的研究

Abstract

經過定點突變的技術，可以將野生型肌紅蛋白His-64、Val-68、Ile-107 分別突變成Asp、Leu、Met。研究指出，經突變的肌紅蛋白具有下列兩種特性：(1)過氧化酶活性，(2) 能夠穩定帶有自由基的陽離子 (FeIV = O Por+•)，進而延長電荷分離的時間。肌紅蛋白的結構已經藉由高解析X-ray 解出，且基本特性也已分析完成，因此肌紅蛋白很適合用來當作電子轉移以及化學活性分析的模型。為了將野生型及突變型肌紅蛋白(MbWT,MbH64D, MbV68L, MbI107M, MbH64D/V68L, MbH64D/V68L/I107M) 應用在以蛋白質為基礎的光-化學能量轉換的系統中及比較其突變的影響，因此，我們將輔基團 鋅-原紫質 (ZnPP) 以及 鋅-乙炔苯酸紫質(ZnPE1) 重組到肌紅蛋白內。藉由紫外光-可見光光譜及螢光光譜分析其生物物理特性以確定輔基團成功地重組到蛋白質的活性中心，並藉此算出其能隙(energy gap)。此外，我們也經由循環伏安法及微差脈衝伏安法求出重組金屬紫質-肌紅蛋白複合體的HOMO/LUMO 電位。在螢光生命期的比較中發現，將輔基團重組到肌紅蛋白內的生命期比單獨輔基團溶解在四氫呋喃 (THF) 內還要。 以蛋白質為基礎的光化學能量轉換的系統包含重組金屬紫質-肌紅蛋白複合體當做感光酵素、三乙醇胺 (TEA) 當作電子提供者以及菸鹼胺腺呤雙核酸磷酸鹽(NADP+) 當作電子接受者。比較不同輔基團以及突變點的重組金屬紫質-肌紅蛋白複合體中，ZnPE1-MbV68L 具有最好的NADP+ 還原效率 (94.7%) 以及連續照光6 小時的反應速率(0.209 mM/hr)。

After replacing His 64 with Asp, Val 68 with Leu, and Ilu 107 with Met of myoglobin (Mb) via molecular engineering, two properties exist: (1) peroxidase activity and (2) the ferryl porphyrin cation radical (FeIV = O Por+•) stabilization for prolonging charge separation. Because the structure of myoglobin had been successfully solved by high-resolution x-ray crystallography and possess the characterization of availability, it is a suitable model to understand the electron-transfer (ET) reaction as well as the chemical reactivity. In order to apply these proteins into protein-based photo-chemical energy conversion system and compare the mutation effect, various Mb mutants, ranging from mono (MbH64D, MbV68L, MbI107M), double (MbH64D/V68L), and triple (MbH64D/V68L/I107M) mutations were reconstituted with prosthetic groups (ZnPP and ZnPE1) to generate metallo-porphyrin Mb complex. Biophysical properties characterization of the metallo-porphyrin Mb complexes, using UV-Vis and fluorescence spectrometry, validates the successful reconstitution of the prosthetic group inside the active site pocket and energy gap of the reconstituted metallo-porphyrin Mbs. Cyclic voltammetry and differential pulse voltammetry techniques further confirmed the HOMO/LUMO of the reconstituted metallo-porphyrin Mbs. In the picosecond fluorescence decays also showed apo-Mb reconstituted with porphyrin prolonged the lifetime compared with porphyrin in THF. The protein-based photo-chemical energy conversion systems were constructed, using mutated apo-Mb reconstituted with different metalloporphyrin-derivatives as photosensitizers, triethanolamine (TEA) as electron donor and nicotinamide adenine dinucleotide phosphate (NADP+) as an electron acceptor. Among different reconstituted metalloporphyrin-Mbs in photoirradiation systems, MbV68L reconstituted with ZnPE1 exhibited the best energy conversion efficiency (94.7 %) and reaction rate within 6 hours photoirradiation (0.209 mM/hr), as comparable to that of the myoglobin reconstituted with ZnPP in photo-chemical energy conversion system.