CYTOCHROME B5--A CASE STUDY IN MOLECULAR RECOGNITION

细胞色素 B5——分子识别案例研究

• 批准号: 2685028
• 负责人: Mario Rivera
• 金额: $ 10.4万
• 依托单位: OKLAHOMA STATE UNIVERSITY STILLWATER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2685028
• 关键词: NAD(P)H oxidoreductase; X ray crystallography; active sites; cadmium; calcium; carbon; cytochrome P450; cytochrome b5 reductase; divalent metal; electrochemistry; erythrocytes; heme; hydrogen bond; intermolecular interaction; liver cells; magnesium; microsomes; mitochondrial membrane; nuclear magnetic resonance spectroscopy; propionates; protein structure function; site directed mutagenesis; stable isotope

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.

细胞色素 b 是参与电子传递的一类重要的血红素蛋白 转移反应。 前者的功能主要是作为一个必要的 脂肪酸去饱和途径中的电子传递成分， 胆固醇生物合成和药物代谢。 找到的表格在 红细胞参与高铁血红蛋白还原途径。 一个 该途径的缺陷使生物体易患高铁血红蛋白血症， 一种病理状况，可导致系统从发绀到精神错乱 迟缓。 我们合成并过度表达了一个编码的基因 来自大鼠肝脏的线粒体外膜细胞色素b5，最近 表明其 E 1\2 比该值负约 100 mV 观察微粒体或红细胞蛋白。 有趣的是，E NADH-细胞色素b5还原酶的1/2为-88 mV，存在时为-102 mV NAD。 这表明线粒体蛋白可能会发生反应 与典型途径的 NADH-细胞色素 b5 还原酶不同 上面提到过。 PI 打算实现以下目标： 1. 制定方法论 通过结合现在的 13C-血红素富集 b5 的细菌表达 阐明了血红素的生物合成途径及其内置的特殊性质 我们的线粒体细胞色素 b5 表达系统。 表达 细胞色素 b5 基因开启血红素合成，然后将其掺入 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 PI，但它对其他感兴趣的研究人员也很有用 血红素蛋白的 NMR，因为同位素标记的血红素可以被去除 从细胞色素 b5 中提取，以便用它重组其他蛋白质，或者 将其用于模型化合物研究。 二. 阐明血红素的作用 细胞色素 b5 中的丙酸盐在与生理伴侣的结合中发挥作用 蛋白质，例如细胞色素 c 和 P-450。 为此，细胞色素 b5 带有13C标记的血红素将用于提取结合等信息 位点、化学计量和结合常数，这对于 了解电子转移反应。 b5 形成的复合物 普遍存在的 Ca2= 和 Mg2+ 离子，最近与 通过与暴露的血红素结合来调节 b5 的 E 1/2 值 丙酸盐，也将通过 13C 和 113 Cd NMR 光谱进行研究。 三． 使用定点诱变、NMR 光谱 X 射线 晶体学和电化学技术来研究主要因素 据信控制双组氨酸连接细胞色素的还原电位 b：这些因素是； a) 水对疏水性血红素的可及性 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 轴向组氨酰咪唑 Ndelta，这会导致更强或更弱 Fe-N键。 d) 轴向组氨酰咪唑平面的几何排列 这可能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 双组氨酸连接的细胞色素 b 中的结构功能以及对研究人员的影响 对分子“模型”领域感兴趣18,19，一类新颖的 参与氧化还原催化的金属蛋白的简化版本 能量转换。