利用超快雷射時間解析光譜研究一氧化氮合成酶之超快動力學

Abstract

一氧化氮（NO）在哺乳動物中具有重要功能：血管穩定與神經傳遞。在哺乳動物體中，一氧化氮可經由一氧化氮合酶（Nitric Oxide Synthase, NOS）合成，一氧化氮合酶在人體中具有三種異構體，依照存在於人體中的位置分為：神經元型（neuronal），內皮型（endothelial）和誘導型（inducible）。其單體都含有C-末端還原酶區 (C-terminal reductase domain) 和N-末端氧化酶區 (N-terminal oxygenase domain)透過鈣調素（Calmodulin, CaM）連接。

鑑於一氧化氮合成酶的生物重要性，因此，一氧化氮合成梅與其相關的蛋白質已廣泛被許多研究團隊研究。其中，時間解析的光譜研究，因能瞭解一氧化氮合成酶的超快動力學特性，雖已有部分團隊研究過，然而，其研究結果僅限於少部分的可見光波長或是波段，對於明白一氧化氮合成酶的超快動力學特性仍顯不足。另外，NOS反應週期關鍵機制為NOS蛋白從NOS氧化酶區NO解離和復合。承上，本研究將以超快時間解析光譜技術，並以紫外光超短脈衝激發，搭配紫外光與可見光的寬頻短脈衝探測技術，藉此闡述一氧化氮合成酶的超快動力學。

我們建構一套短脈衝雷射紫外光激發與多波段探測的時間解析光譜技術，為了確保樣品不受雷射激發而損害，量測時需縮短時間解析光譜實驗數據取樣的時間，因此，我們建構寬頻譜快速掃描與取樣技術。另外，為能妥善分析時間解析光譜數據，我們使用全域擬合分析方式(Global fitting analysis)以及二維相關連光譜分析技術(Two-dimensional correlation spectroscopy)，對於實驗數據進行分析。在本研究中，使用的一氧化氮合成酶為內皮型NOS（eNOS）氧化區域 (oxygenase domain)，簡稱：eNOS-oxy。

在第三章，我們以紫外光短脈衝雷射激發Soret band來探討Imidazole 與 L-arginine對內皮型NOS（eNOS）的作用。雷射光激發eNOS導致Imidazole與原血紅素(Heme)的鍵結斷裂(ligand dissociation)，其斷裂時間發生在小於0.3 皮秒內，緊接著在2皮秒內發生震動冷卻(vibrational cooling)以及Imidazole再復合 (ligand recombination). 光激發導致鍵結斷裂與再鍵結過程也產生蛋白質內振盪(protein quakes), 此現象在數十皮秒內完成。eNOS-oxy與L-arginine在光激發下，其動力學機制主要為內部能量轉換(約0.4皮秒)，接著為振動冷卻、電子傳遞以及基態恢復等事件發生於1.4皮秒內。就eNOS-oxy而言，在沒有接上Imidazole或L-arginine時，其內部的原血紅素與水分子鍵結，此鍵結為部分鍵結(partially bonding)，因此，在光激發後的動力學為：在0.8皮秒內發生鍵結斷裂，接續為1.4皮秒內發生震動冷卻並伴隨電子轉移，最後為水分子與原血紅素產生部份再次鍵結的過程，其過程發生在12皮秒內。

在第四章，我們用超快時間解析紫外光激發探測光譜技術研究eNOS-oxy內原血紅素的光致自旋態轉變(photo-induced spin state transition)。透過靜態吸收光譜擬合分析以及動態時間解析光譜的擬合分析，我們得到光致自旋態遷移的比例與動態時間解析光譜的訊號具有關聯性。

在第五章，我們以紫外光(400 nm) 激發與寬頻可見光(450 nm~700 nm) 短脈衝雷射時間解析光譜探討eNOS-oxy的動力學，其探測光波段涵蓋eNOS-oxy吸收光譜Q頻帶 (Q-band)與電子轉移頻帶 (charge transfer band)，並用全域擬合分析方式(global fitting analysis) 以及二維相關聯光譜分析(two-dimensional correlation spectroscopy)，我們得到其動力學機制如下：電子由Soret band轉移至Q-band時間為0.16 皮秒，水分子鍵結斷裂伴隨電子傳遞時間為0.94 皮秒，電子轉移態的鬆弛時間為4.0皮秒，水分子再鍵結的時間為59皮秒。

總結本研究，我們利用自行建構的超快時間解析光譜技術研究eNOS-oxy，因其具有多波段且短脈衝特性，加上具有快速擷取數據的能力，可避免雷射的不穩定性以及樣品因長時間受雷射照射而破壞的問題。我們提出一系列的實驗結果，說明eNOS-oxy的超快動力學機制。研究結果，預計能對NOS有利於人體健康的藥物干預工作給予相關的知識。

Nitric oxide (NO) has important functions in mammals to control a plethora of cellular activities including vascular homeostasis, neurotransmission, and host defense. The NO can be biosynthesized by three isoforms of nitric oxide synthases (NOS), neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All three NOS isomers are homodimer in which each monomer contains a C-terminal reductase domain and an N-terminal oxygenase domain connected to each other via calmoduline (CaM)-biding sequence. NO can be released from heme in the oxygenase domain after electron transfer from the reductase domain to the oxygenase domain. Because of the biological importance of the NOS, NOS and NOS-like proteins have been widely studied in various research groups. Time-resolved absorption change measurements at a few probe wavelengths are still not sufficient to clarify the dynamics. In the present work, we elucidate the mechanism of the NOS protein dynamics starting from observation of the dissociation and recombination of NO in oxygenase domain of NOS, which is key reaction in the reaction cycle of NOS

Ultrafast transient absorption spectroscopy of endothelial NOS oxygenase domain (eNOS-oxy) was performed to study dynamics of ligand or substrate interaction under Soret band excitation (see chapter 3). Photo-excitation dissociates imidazole ligand in less than 300fs, and then followed by vibrational cooling and recombination within 2 ps. Such impulsive bond breaking and late rebinding generate proteinquakes, which relaxes in several tens of picoseconds. The photo-excited dynamics of eNOS-oxy with L-arginine (L-Arg) substrate mainly occurs at the local site of heme, including ultrafast internal conversion within 400 fs, vibrational cooling, charge transfer, and complete ground-state recovery within 1.4 ps. The eNOS-oxy without additive is partially bound with water molecule, thus its photoexcited dynamics also shows ligand dissociation in less than 800 fs. Then it followed by vibrational cooling coupled with charge transfer in 4.8 ps, and recombination of ligand to distal side of heme in 12 ps

Photo-induced spin state transition of ligand substrate interaction of endothelial NOS oxygenase domain (eNOS-oxy) was studied by ultrafast transient absorption spectroscopy under Soret band excitation (see chapter 4). We have demonstrated the spectral analyze method, which combined TA spectra and steady absorption spectra to study the percentage of photo-induced spin state transition in three case i.e. eNOS-oxy, eNOS-oxy/Im, and eNOS-oxy/L-Arg.

Ultrafast dynamics of eNOS oxygenase domain was studied by transient absorption spectroscopy pumping at Soret band (see chapter 5). The broadband visible probe spectrum has visualized the relaxation dynamics from the Soret band to Q-band and charge transfer (CT) band. Supported by two-dimensional correlation spectroscopy, global fitting analysis concludes the relaxation dynamics from the Soret band as (1) electronic transition to Q-band (0.16 ps), (2) ligand dissociation and CT (0.94 ps), (3) relaxation of the CT state (4.0 ps), and (4) ligand rebinding (59 ps).

We reported a comprehensive investigation of the ultrafast dynamics in heme of eNOS-oxy domain. These are expected to give key knowledge for future work on pharmacological intervention against individual isoforms of NOS contributing to human health.