細胞色素C是一種電子攜帶蛋白，在電子傳遞鍊中會從cytochrome c reductase接受電子後再將電子傳遞給cytochrome c oxidase，除了電子傳遞的生理功能之外，細胞色素C具有促進Apaf-1與pro-caspase-9聚集形成apoptosome的複合體而導致細胞走向細胞凋零及促進氧化壓力(oxidative stress)誘導疾病發生之生理功能，細胞色素C能具有多樣性的生理功能，主要都是必須與其他的蛋白質進行蛋白質與蛋白質間交互作用(protein-protein interaction)，才能執行正常的生理功能；雖然，已經有許多物種的細胞色素C被純化出來並做過許多詳細的研究，但是人類的細胞色素C的相關研究則相當貧乏，為了更透徹地研究人類細胞色素C，使人類細胞色素C可作為治療用蛋白，以及探討細胞色素C與Apaf-1如何進行交互作用及細胞色素C被nitrosylation後對其結構之影響，因此，首先我們必須獲得具有正確結構與功能的人類細胞色素C重組蛋白。

　　微過氧化酶為水溶性的微血基質蛋白質，是一個被廣泛使用的血基質蛋白質模型，含有不同胜肽數的微過氧化酶(MP-5, MP-6, MP-8, MP-9, & MP-11)可由細胞色素C經不同的蛋白水解酶的水解後獲得，而乙醯化後的微過氧化酶(Ac-MPs)更可提高其對水的溶解度，微過氧化酶本身具有過氧化酶的酵素活性，在含有過氧化氫的狀態下可經由過氧化酶型態的反應氧化許多的有機物，除了過氧化酶的酵素活性之外，微過氧化酶也具有cytochrome P-450即單氧化酶(monooxygenase)的酵素活性可以對許多的有機物進行氫氧化反應，然而，微過氧化酶的過氧化氫酶活性尚未被清楚的研究，以及微過氧化酶的酵素活性比天然的過氧化酶的酵素活性低很多，因此，我們則對微過氧化酶的過氧化氫酶活性作了進一步的分析，以及為了改善微過氧化酶的酵素活性，我們利用蛋白質基因工程的方式製備不同的酵母菌細胞色素C的突變株，進而製備出各種不同的微過氧化酶進行相關的研究。

　　在人類細胞色素C的研究中，我們在大腸桿菌中成功地表現出及純化到10-15 mg/L的人類細胞色素C重組蛋白，在氧化還原電位分析中，我們所製備的人類細胞色素C重組蛋白為0.246伏特與天然馬類細胞色素C的0.249伏特有近似的結果，同時根據NMR的研究結果發現人類細胞色素C重組蛋白與天然馬類細胞色素C具有相同的三級結構，進一步為了研究細胞色素C與Apaf-1如何進行交互作用，我們利用結構模擬的方式建構Apaf-1的兩個WD-40 repeats區域的結構，然後蛋白質docking的方式，推測出細胞色素C與Apaf-1進行交互作用的方式，我們的結果不僅證實利用大腸桿菌表現出來的人類細胞色素C重組蛋白與天然馬類細胞色素C具有相似的功能與結構，而且目前已經知道細胞色素C與許多的疾病的發生有關，因此，針對細胞色素C以及與細胞色素C有交互作用之蛋白的功能與結構之研究，將有助於獲得及洞悉與人類疾病相關資訊之基礎。

　　在微過氧化酶的研究中，我們發現微過氧化酶除了已知的過氧化酶與單氧化酶二種酵素型態的活性以外還具有過氧化氫酶型態的酵素活性，其中Ac-MP-8分解過氧化氫時的Km與kcat分別為40.9 mM與4.1 sec^-1，而Ac-MP-8對分解過氧化氫的酵素活性(specificity constant)(kcat/Km)為100.2 M^-1 sec^-1，這結果分別為過氧化酶酵素型態的活性的1/12 ~ 1/5及單氧化酶酵素型態的活性的1/100 ~ 1/50，這結果顯示微過氧化酶具有過氧化酶、單氧化酶及過氧化氫酶這三種酵素型態的活性，而第五配位基為組氨酸(Histidine)的微過氧化酶在這三種酵素型態的活性高低分別為：過氧化酶＞單氧化酶＞過氧化氫酶，在微過氧化酶進行過氧化酶酵素活性測定中加入一些guanidinium hydrochoride或ammonium-derivative bases分子，如： Mes、Mops、Hepes、Tricine與Tris，可以增加Ac-MP-8的過氧化酶型態的酵素活性3 ~ 11倍，另外，四種利用酵母菌細胞色素C的突變株所製備出來的微過氧化酶實驗中，我們發現在Cys-14到Cys-17中的胺基酸置換成帶正電荷的離氨酸(lysine)或蛋氨酸(arginine)可以增加其過氧化酶型態的酵素活性至原先活性的兩倍，此外，因為微過氧化酶在作為生物感應器(biosensor)及工業應用上具有相當大的發展潛力，因此，我們研究的成果希望可作為改善微過氧化酶酵素活性的基礎，用以發展出高活性之微過氧化酶期望可作為工業應用之酵素。

　　Cytochrome c is an electron carrier protein that is responsible for accepting an electron from cytochrome c reductase and for transferring an electron to cytochrome c oxidase. In addition to functioning as an electron carrier, cytochrome c promotes the assembly of a caspase-activating complex to induce cell apoptosis and stimulates the oxidative stress-induced diseases. These diverse functions of cytochrome c are associated with the protein-protein interactions between cytochrome c and its interacting proteins. Although many cytochromes c from different species have been purified and studied, little is known about human cytochrome c. To characterize human cytochrome c, to use human cytochrome c as the therapeutic agent, and to study how cytochrome c interacts with Apaf-1 and effect of nitrosylation on structure-activity relationships of cytochrome c, it is essential to obtain recombinant human cytochrome c with the correct fold.

　　Microperoxidases (MPs) are water-soluble heme species that can serve as models for the heme proteins. MPs with various peptides (MP-5, MP-6, MP-8, MP-9, MP-11, & MP-17) are obtained by proteolytic digestion of cytochromes c. (Ac-MPs) exhibit much less tendency to aggregate in aqueous solution than their unacetylated precursor. Both MPs and Ac-MPs the peroxidase activities and can convert a wide variety of organic compounds at the expense of hydrogen peroxide in a peroxidase-type reaction. In addition to the peroxidase-type reaction, MP also catalyzes cytochrome P450-type oxygen-transfer reactions. However, the catalase activity of MP is not well understood and the enzyme activities of MP are much lower than native enzyme. To improve the enzyme activities of MP, we engineered yeast cytochrome c by site-directed mutagenesis and obtained the modified MPs by proteolytic digestion of recombinant cytochromes c.

　　In this study of human cytochrome c, we expressed human cytochrome c in E. coli and purified to homogeneity with a yield of 10-15 mg/L. The redox potential of recombinant human cytochrome c was 0.246 V which was measured by cyclic volatmmetry measurement. This is similar to that of horse cytochrome c with a value of 0.249 V. Based on our NMR studies, the recombinant human cytochrome c produced in E. coli exhibits the same tertiary fold as horse cytochrome c. To study the interaction between human cytochrome c and Apaf-1, we modeled 3D structure of Apaf-1 by homology modeling and docked the structure of cytochrome c into Apaf-1 structure. Our results provided the evidence that human cytochrome c expressed in E. coli possesses similar function and structure as that of horse protein. It is known that cytochrome c plays a role in many human diseases. This study serves as the basis for gaining insight into human diseases by exploring structure and function relationships of cytochrome c to its interacting proteins.

　　In the study of microperoxidase, we report here that Ac-MP-8 catalyzes catalase-type reaction in addition to peroxidase-type and cytochrome P450-type reactions. The Km and kcat of the decomposition of H2O2 catalyzed by Ac-MP-8 are 40.9 mM and 4.1 sec^-1, respectively. The specificity constant (kcat/Km) of Ac-MP-8 in catalase-type reaction of H2O2 is 100.2 M^-1 sec^-1 which is 5- to 12- and 50- to 100-fold less than those of microperoxidases in cytochrome P450-type reaction of aniline/H2O2 and peroxidase-type reaction of o-methoxyphenol/H2O2, respectively. These results indicate that Ac-MP-8 can catalyze three different types of reactions, and the relative catalytic specificities of Ac-MP-8 with a histidyl ligand exhibit the following orders: peroxidase-type > cytochrome P450-type > catalase-type reactions. Kinetic studies on Ac-MP-8 showed that guanidinium hydrochloride and ammonium-derivative based (MES, MOPS, HEPES, Tricine, and Tris) can cause 3-11 folds increase in the peroxidase activity of Ac-MP-8. Four modified MPs were obtained by proteolytic digestion of recombinant yeast cytochromes c. The mutation of the residues between Cys-14 and Cys-17 to the R or K residues caused 2-fold increase in peroxidase activity. It is known that MP has potential for application to biosensor technology. This study serves as the basis to improve the enzyme activities of MP that can be used for industrial application.

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