CYTOCHROME B5--A CASE STUDY IN MOLECULAR RECOGNITION

细胞色素B是一类重要的血红素蛋白， 转移反应 前者主要是作为一种基本的 脂肪酸去饱和途径中的电子转移组分， 胆固醇生物合成和药物代谢。 形式在 红细胞参与高铁血红蛋白还原的途径。 一 该途径的缺乏使生物体易患高铁血红蛋白血症， 一种病理状态，可导致系统从发绀到精神 迟钝 我们合成并过度表达了一种基因， 大鼠肝脏线粒体外膜细胞色素b5，最近 显示其E1\2比 观察微粒体或红细胞蛋白。 有趣的是，E 1/2的NADH-细胞色素b5还原酶为-88 mV， 的NAD。 这表明线粒体蛋白可能会对 与典型途径的NADH-细胞色素b5还原酶不同， 上面提到的。 主要研究者计划实现以下目标：1. 制定方法 通过结合现在的13 C-血红素富集的b5的细菌表达， 阐明了血红素的生物合成途径和特殊性质， 我们的线粒体细胞色素b5的表达系统。 表达 细胞色素b5基因启动血红素合成， 在过表达的多肽中，从而简化了分离和 血红素的纯化。 这种方法不仅有利于研究 但它也将是有用的其他研究人员感兴趣的， 血红素蛋白质的NMR，因为同位素标记的血红素可以被去除 从细胞色素b5中分离出来，以便用它重建其他蛋白质，或者 在模型化合物研究中使用它。 二. 阐明血红素 细胞色素b5中丙酸盐在与生理伴侣结合中起作用 细胞色素c和P-450等蛋白质。 为此，细胞色素b5 13 C标记的血红素将用于提取信息，如结合 位点，化学计量和结合常数，这将是有用的 了解电子转移反应。 b5形成的复合物 普遍存在的Ca 2+和Mg 2+离子，最近被牵连在 通过与暴露的血红素结合来调节b5的E1/2值 丙酸盐，也将通过13 C和113 Cd NMR光谱进行研究。 三. 使用定点诱变，核磁共振光谱X射线 晶体学和电化学技术来研究主要因素 据信控制双-His连接的细胞色素的还原电位 B：这些因素是：a）水对疏水血红素的可及性 环境 B）带电残基之间的库仑相互作用关闭 血红素和铁血红素上的正电荷。 C.质子化程度 的轴向组氨酰咪唑Ndelta，这导致在一个更强或更弱的 Fe-N键 d）轴向组氨酰咪唑平面的几何排列 这可能会受到轴向周围氢键网络的影响 配体。 这些信息将有助于详细了解 在双组氨酸连接的细胞色素B结构功能，并向研究人员 感兴趣的领域的分子“maquettes“18，19，一类新的 简化版本的金属蛋白参与氧化还原催化和 能量转换

Cytochromes b are an important class of hemoproteins involved in electron transfer reactions. The former functions primarily as an essential electron transfer component in the pathways of fatty acid desaturation, cholesterol biosynthesis and drug metabolism. The form found in erythrocytes is involved in the pathway for methemoglobin reduction. A deficiency in this pathway predisposes the organism to methemoglobinemia, a pathological condition which can cause systems from cyanosis to mental retardation. We have synthesized and overexpressed a gene that codes for the outer mitochondrial membrane cytochrome b5 from rat liver, and recently shown that its E 1\2 is approximately 100 mV more negative than that observed for the microsomal or erythrocyte proteins. Interestingly, the E 1/2 of NADH-cytochrome b5 reductase is -88 mV, and -102 mV in the presence of NAD. This suggests that the mitochondrial protein is likely to react differently with the NADH-cytochrome b5 reductase typical of the pathways mentioned above. The PI intends to accomplish the following; 1. Develop the methodology for the bacterial expression of 13C-heme enriched b5 by combining the now elucidated biosynthetic pathway of heme and the special properties built in our expression system of the mitochondrial cytochrome b5. Expression of the cytochrome b5 gene turns on heme synthesis, which is then incorporated in the overexpressed polypeptide, thus simplifying the isolation and purification of heme. This methodology will not only benefit the research of the PI, but it will also be useful to other researchers interested in NMR of heme proteins, since the isotopically labelled heme can be removed from cytochrome b5 in order to reconstitute other proteins with it, or to use it in model compound studies. II. Elucidate the role that heme propionates in cytochrome b5 play in binding to physiological partner proteins such as cytochromes c and P-450. To these ends, cytochrome b5 with 13C labelled heme will be used to extract information such as binding sites, stoichiometries and binding constants, which will be useful for the understanding of electron transfer reactions. The complexes that b5 forms with ubiquitous Ca2= and Mg2+ ions, which have recently been implicated in modulating the E 1/2 value of b5, by binding to the exposed heme propionates, will also be studied by 13C and 113 Cd NMR spectroscopies. III. Use site directed mutagenesis, NMR spectroscopy X-ray crystallographic and electrochemical techniques to study the major factors believed to control the reduction potential of bis-His ligated cytochromes b: These factors are; a) Accessibility of water to the hydrophobic heme environment. b) Coulombic interactions between charged residues close the heme and the positive charge on the ferric heme. c) Degree of protonation of axial histidyl imidazole Ndelta, which results in a stronger or weaker Fe-N bond. d) Geometrical arrangement of axial histidyl imidazole planes which can be influenced by the hydrogen bond network around the axial ligand. This information will be useful for the detailed understanding of structure function in the bis-His ligated cytochromes b, and to researchers interested in the area of molecular "maquettes "18,19, a novel class of simplified versions of metalloproteins involved in redox catalysis and in energy conversion.