CYTOCHROME B5--A CASE STUDY IN MOLECULAR RECOGNITION

细胞色素 B5——分子识别案例研究

• 批准号: 2392193
• 负责人: Mario Rivera
• 金额: $ 10.08万
• 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/2392193
• 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是与电子有关的重要类血蛋白 转移反应。 前者主要是必不可少的 脂肪酸去饱和途径中的电子转移成分， 胆固醇生物合成和药物代谢。 在 红细胞参与了低铁蛋白还原的途径。 一个 这种途径中的缺乏使生物体易受非生物血红素血症， 一种病理状况，可能导致从氰化物到精神的系统 迟钝。 我们已经合成并过表达编码的基因 大鼠肝脏的线粒体膜细胞色素B5，最近 表明其E 1 \ 2的负值约为100 mV 观察到微粒体或红细胞蛋白。 有趣的是，e NADH -CytoChrome B5还原酶的1/2为-88 mV，在存在下-102 mV 纳德。 这表明线粒体蛋白可能反应 与典型的途径的NADH-CYTOCHROME B5还原酶不同的方式不同 上面提到的。 PI打算完成以下内容； 1。开发方法 通过结合现在的细菌表达13C-血红素富集B5 阐明血红素的生物合成途径和内置的特殊特性 我们的线粒体细胞色素B5的表达系统。 表达 细胞色素b5基因打开血红素的合成，然后将其掺入 在过表达的多肽中，从而简化了隔离和 血红素的纯化。 这种方法不仅将使研究受益 PI的，但也对其他有兴趣的研究人员很有用 血红素蛋白的NMR，因为可以去除同位素标记的血红素 从细胞色素B5来重新构建其他蛋白质，或 在模型复合研究中使用它。 ii。 阐明血红素的作用 细胞色素B5中的丙酸 细胞色素C和P-450等蛋白质。 到这些目的，细胞色素b5 带有13C标记的血红素的血红素将用于提取诸如绑定之类的信息 位点，化学计量法和结合常数，这将对 了解电子转移反应。 B5形成的复合物 使用无处不在的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 丙酸酯也将通过13C和113 cd NMR光谱研究来研究。 iii。 使用定向诱变，NMR光谱X射线 晶体学和电化学技术研究主要因素 认为可以控制双his连接的细胞色素的降低潜力 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 血红素和铁血红素的阳性电荷。 c）质子化程度 轴向组咪基咪唑ndelta的含量，导致更强或较弱 Fe-n债券。 d）轴向组咪基平面的几何布置 它可以受轴向周围的氢键网络的影响 配体。 此信息对于详细了解 BIS-HIS结扎的细胞色素B和研究人员的结构功能 对分子“ maquettes”领域感兴趣18,19，这是一种新颖的类 与氧化还原催化有关的金属蛋白的简化版本和 能量转换。