细胞色素c向过氧化物酶结构—功能的转化研究

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作者：王中华 论文级别：博士 学科专业名称：无机化学 中文关键词：细胞色素c ; 血红素蛋白 ; 过氧化物酶 ; 分子设计 ; 结构—功能转化 英文关键词：Cytochrome c ; hemoprotein ; peroxidase ; molecular design ; structure-function conversion 学位年度：2007 导师：黄仲贤 学科代码：070301 学位授予单位：复旦大学 论文提交日期：2007-04-15

摘要

血红素蛋白是一大类含有原卟啉Ⅸ(血红素，heme)作为辅基的金属蛋白，在生命体系中执行着重要的生物功能，如：血红蛋白(hemoglobin，Hb)和肌红蛋白(myoglobin，Mb)等具有载氧功能，细胞色素c(cytochrome c，Cyt c)和细胞色素b_5(cytochrome b_5，Cyt b_5)等具有电子传递功能，细胞色素P450(cytochrome P450，Cyt P450)和细胞色素c过氧化物酶(cytochrome c peroxidase，CcP)等具有生物催化功能，CO敏感器(CooA)和NO敏感器(soluble guanylate cyclase，sGC)具有生物传感功能等。相同的辅基heme如何被不同的蛋白分子所利用，来执行不同的生物功能，一直是化学生物学和蛋白质化学研究领域所关注的重点，人们正试图对这一问题进行解答来认识金属蛋白结构—性质—反应—功能之间所蕴含的微妙关系。
     Cyt c是血红素蛋白中电子传递蛋白的典型代表，其辅基heme与蛋白肽链的结合除了来自heme轴向配体组氨酸(His18)和甲硫氨酸(Met80)的配位作用、氢键和疏水相互作用等非共价键作用方式外，血红辅基heme的两个乙烯基还与蛋白肽链上的结构片段域Cys-Xaa-Xaa-Cys-His(CXXCH)中的两个半胱氨酸形成硫醚键以共价键方式相结合。因而Cyt c的血红素辅基(heme c)与蛋白多肽链之间的结合很牢固，蛋白质的性质相对很稳定。
     正是由于Cyt c蛋白的稳定性使其不但是血红素蛋白结构与功能关系研究的典型代表，而且也是血红素蛋白结构与功能转换研究的良好模板蛋白。虽然Cyt c在生物体内的主要功能是在线粒体中传递电子和参与细胞调亡，在生物体外的一定条件下却也能表现出过氧化物酶活性并具有诸多优点，如：抗热变性、抗有机溶剂等。但与辣根过氧化物酶(horseradish peroxidase，HRP)和细胞色素c过氧化物酶等天然过氧化物酶相比，Cyt c的过氧化物酶活性是很低的，其原因主要在于Cyt c与HRP及CcP等天然过氧化物酶在结构上存在较大的差异。通过晶体结构对比分析，我们不难发现Cyt c与HRP的结构之间主要存在以下区别：首先是血红素辅基heme在HRP中是处于五配位高自旋状态，而在Cyt c中是处于六配位低自旋；其次是HRP中heme的轴向位置有一个组氨酸，但没有与heme配位，而是一个远端组氨酸(distal histidine)；除此之外，HRP还有一个带正电荷的远端精氨酸(distal arginine)存在，这些结构特征都是Cyt c所不具备的。HRP所具有的这些结构特点不但有利于过氧化物酶反应过程中活性中间体CompoundⅠ的形成，而且也有利于反应底物与酶的结合。
     虽然对Cyt c蛋白分子进行定点突变及其过氧化物酶活性研究的报道不少，但却几乎没有人对如何将Cyt c转化成为类过氧化物酶(peroxidase-like)分子进行过专门的分子设计。因此通过分子设计和蛋白质工程等方法，将Cyt c这个具有较高稳定性的电子传递蛋白转化为一个具有较高pemxidase-like活性的酶分子，不但有助于我们对血红素蛋白结构—性质—反应—功能之间关系的理解，同时也有可能创造出一个具有一定应用前景的、新的金属蛋白酶。
     通过对Cyt c的晶体结构进行观察分析后，我们发现Pro71几乎位于heme辅基Met80一侧的正上方，如果将Pro71替换成His71后，His71上的Nε原子与heme中心Fe~(3+)间的距离为～5.62(?)，非常接近HRP和CcP分子中远端组氨酸Nε原子到heme中心Fe~(3+)的距离(在HRP和CcP中该距离分别为～5.84和～5.55(?))。因而我们通过基因突变方法，首先用一个无配位能力的缬氨酸(Valine，Va1)取代了轴向配体Met80，得到一个Cyt c的单点突变体(Cyt c M80V，M80V)；在此基础上又用histidine取代了71位的proline，得到一个双点突变体(Cyt cP71H／M80V，P71H／M80V)。突变体蛋白的peroxidase-like催化活性实验结果显示，Cyt c M80V突变体蛋白的peroxidase-like催化活性确实比野生型细胞色素c(wild type cytochrome c，WT Cyt c)高；然而，再在71位引入一个“distal histidine”后所得Cyt c P71H／M80V双点突变体蛋白的催化活性不仅没有比Cyt c M80V高，反而比WT Cyt c还低。于是我们对P71H／M80V蛋白性质进行了一系列的研究，结果表明可能是由于Cyt c中Ωloop D肽段(residues 70-84)具有很强的柔韧性，使得His71并没有象HRP中的distal histidine一样起到酸碱催化功能，而是与heme发生了配位作用，无论是在氧化态还是还原态都成了heme的第六轴向配体。
     由于在71位引入的histidine没有起到distal histidine的作用，我们对Cyt c分子再次进行分析后发现，Tyr67虽然不象Pro71一样位于heme的正上方(假定heme位于水平面上，第五轴向配体His18位于heme的下方)，但也位于heme的斜上方。同时分子模拟结果显示，如果将Tyr67突变成His67后，His67的Nε原子与heme中心Fe~(3+)间的距离为～5.18(?)，接近HRP中distal histidine到heme中心Fe~(3+)的距离(～5.84(?))，于是我们构建了Cyt c Y67H和Y67H／M80V突变体基因并进行了蛋白表达和性质研究。研究结果表明，Cyt c Y67H和Y67H／M80V蛋白的过氧化物酶活性都在WT Cyt c的基础上得到了很大的提高；而且在同样条件下，Cyt c Y67H催化氧化邻甲氧基苯酚反应得到的表观二级反应速率常数(k_(cat)／K_m)比天然过氧化物酶HRP还高。
     在天然过氧化物酶(如HRP)中除了有一个远端组氨酸外，还有一个远端精氨酸(distal arginine)。为了模拟过氧化物酶的这一结构，我们又制备了Cyt cY67R和Y67R／P71H／M80V突变体蛋白，并对它们的基本性质和催化活性进行了研究。结果表明在Cyt c的heme腔中引入的arginine(Arg67)确实提高了Cyt c的催化活性，但同时也可能是由于Arg的R基团较大而产生的空间效应影响了底物(guaiacol)的结合，从而导致了米氏常数(K_m)的增加。
     此外，为了进一步研究在heme的轴向配体Met80存在的情况下，Cyt c中第71位的保守氨基酸残基proline被具有较强配位能力的histidine替换后，对Cyt c蛋白结构和性质的影响，我们又制备了Cyt c P71H这个单点突变体蛋白。通过一系列光谱学性质研究后，我们发现在Cyt c P71H中heme的轴向配体随蛋白所处的氧化还原状态不同而发生变化，在氧化态时heme的第六轴向配体是His71，而在还原态时和WT Cyt c一样仍然是Met80。这一研究表明Cyt c分子中位于Ωloop D肽段中的Pro71对稳定Cyt c分子的构象有着重要的作用，在加上Ωloop D这一肽段本身就比较柔韧，所以要想在71位构建一个distal histidine需要重新进行分子设计。
     总之，本论文的这些研究不但丰富了我们对Cyt c分子的认识，同时也进一步拓宽了我们对血红素蛋白结构—性质—反应—功能关系的理解。
Hemoproteins, a large class of metalloproteins which have iron protohemeⅨ(heme) as a common prosthetic group, play versatile roles in biological systems, such as oxygen transport (e.g. hemoglobin, Hb and myoglobin, Mb), electron transfer (e.g. cytochrome c, Cyt c and cytochrome b_5, Cyt b_5) , biological catalysis (e.g. cytochrome P450, Cyt P450 and cytochrome c peroxidase, CcP) and biological sensor (e.g. CooA for CO and sGC for NO). By sharing the same prosthetic group, how various hemoproteins can perform a range of functions by means of different combination of heme and protein scaffolds is a major subject of chemical biology and protein chemistry. People, including chemists and biologists, are attempting to answer such a question so as to understand the precise structure-property-reactivity-function relationship of metalloproteins.
     Cyt c is a typical electron-transfer hemoprotein. In addition to the two axial ligations provided by His18 and Met80, the hydrogen bond and hydrophobic interactions between the heme and the protein polypeptide, the heme also covalently linked to the protein matrix through two thioether bonds which are formed between vinyl groups of the heme and cysteine residues of a classic Cys-Xaa-Xaa-Cys-His (CXXCH) heme-binding motif of the polypeptide chain. Therefore, Cyt c is a very stable protein.
     It is the stability of Cyt c that makes it not only the typical representative for studying the structure-function relationship, but also an excellent template for structure-function convention study of hemoproteins. In living systems, the main functions of Cyt c are electron transfer and inducing apoptosis, but Cyt c, under certain conditions, can also catalyze peroxidase-like reactions and presents several advantages. For example, Cyt c is able to perform catalytic reactions at high temperature and high concentration of organic solvents. However, compared with natural peroxidase, such as horseradish peroxidase (HRP) and cytochrome c peroxidase (CcP), the peroxidase activity of Cyt c is very low because of the differences of their structures. After careful comparison of the structures of Cyt c and HPR, we could find out that there are structural differences between them. HRP has a 5-cooridination high spin heme iron, and a distal histidine as well as a positively charged arginine in the heme pocket, whereas, Cyt c has a 6-coordination low spin heme iron but it lacks the distal histidine as well as the distal arginine in its heme pocket. The structural characters of HRP not only benefit the formation of compound I but also facilitate the access of substrate in the peroxidase reaction cycle.
     Even though many researches on site-directed mutation and peroxidase reaction of Cyt c have been reported, no one has specialized on how to convert Cyt c into a peroxidase-like enzyme by molecular design. Therefore, by means of molecular design and protein engineering, converting Cyt c, a stable electron-transfer hemoprotein, into an efficient, peroxidase-like metalloenzyme can not only help us to understand the structure-property-reaction-function relationship of hemoproteins but also probably create a new metalloenzyme with potential application in industry.
     Based on the crystal structure analysis of Cyt c (PDB ID 2YCC), we found that Pro71 is almost located at the distal position of the heme pocket. If it is replaced by a histidine, the distance between Nεatom of His71 and heme ferric iron is～5．62 ?, which is close to the distance between Nεatom of the distal histidine and heme iron in HRP and CcP (～5．84 and～5．55 ?, respectively). Therefore, we first constructed a single-site mutant (Cyt c M80V, M80V) by replacing the heme axial ligand Met80 with valine that has not coordination capacity, and then we introduced a "distal" histidine at position 71 by constructing a double-site mutant of Cyt c P71H/M80V using site-directed mutagenesis.
     As expected, the peroxidase activity of the Cyt c M80V mutant was enhanced by the vacancy at the sixth ligand site of the heme. In contrast, the introduction of a "distal" histidine in the double-site variant, Cyt c P71H/M80V, did not increase the peroxidase activity at all, but rather caused it to decrease. Therefore, we characterized the Cyt c P71H/M80V variant protein electrochemically and spectroscopically. All the experimental results showed that the introduced histidine at position 71 did not act as a functional distal-histidine, but most likely coordinated to the heme iron, maybe due to the flexibility of theΩloop segment (resides 70-84) of Cyt c protein.
     Because of failed construction of a distal histidine at position 71 in Cyt c, we further examined the crystal structure of Cyt c and found out that Tyr67 is also situated above the heme plane. At the same time, molecular simulation suggests that the replacement of Tyr67 with histidine should place the Nεof His67 at～5．18 ? from the heme iron, a distance that approximates the distance between the Ns of the distal histidine and the heme iron in HRP and CcP (5．84 and 5．55 ?, respectively). Thus we constructed Cyt c Y67H and Y67H/M80V mutant genes and expressed them to proteins. Kinetic studies showed that the obtained Cyt c variants, Cyt c Y67H and Y67H/M80V have much higher peroxidase activities than the wild-type cytochrome protein. Moreover, the apparent second-order rate constant (K_(cat)/K_m) toward guaiacol oxidation of Cyt c Y67H was even higher than that of the native horseradish peroxidase under the same conditions.
     Besides a distal histidine, typical peroxidases, such as HRP and CcP, usually have a distal arginine in the distal heme pocket. In order to mimic this feature we introduced an arginine in the heme pocket of cytochrome c by constructing Cyt c Y67R and Y67R/P71H/M80V variants. The two mutant proteins were expressed, and their peroxidase activities were examined with guaiacol as substrate. The results showed that the introduced arginine indeed enhances the peroxidase activity of Cyt c. However, the Michaelis-Menten constant (K_m) is also increased probably due to the steric effect brought up by the large R-group of arginine which inhibits the substrate access to the heme pocket.
     In order to further examine the effects of Pro71His mutation on the protein structure and property in the presence of Met80, we prepared another mutant of Cyt c, Cyt c P71H variant protein. UV-visible, CD and resonance Raman spectroscopies indicate that in the oxidized mutant the histidine (His71) serves as the sixth axial ligand, while the Met80 still coordinate to the heme in the reduced form. This study illustrates that the Pro71, in theΩloop D segment, plays important roles in stabilizing the structure of Cyt c. So, further molecular designs are required in order to construct a functional distal histidine at position 71 in the heme pocket of Cyt c.
     In summary, the above research results not only enrich our knowledge about Cyt c per se, but also extend our understanding of the structure-property-reactivity-function relationships of other hemoproteins.

引文

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