CYTOCHROME B5--A CASE STUDY IN MOLECULAR RECOGNITION

细胞色素 B5——分子识别案例研究

• 批准号: 2900816
• 负责人: Mario Rivera
• 金额: $ 10.72万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 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 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900816
• 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是一类重要的与电子有关的血红素蛋白。 转移反应。前者的功能主要是作为一个基本的 脂肪酸去饱和途径中的电子转移成分， 胆固醇的生物合成和药物代谢。中找到的表格 红细胞参与高铁血红蛋白还原的途径。一个 这一途径的缺失使机体易患高铁血红蛋白血症， 一种病理状态，可导致从发紫到精神的各种系统 智力迟缓。我们已经合成并过度表达了一种编码 大鼠肝脏线粒体膜外膜细胞色素b5的研究 结果表明，它的E_1\2比它大约负100 mV。 观察微粒体或红细胞的蛋白质。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的成员。这表明线粒体蛋白很可能会发生反应 与典型的NADH-细胞色素b5还原酶不同 如上所述。 PI打算完成以下工作：1.发展方法论 用于富含~(13)C-血红素的b5的细菌表达 阐明了血红素的生物合成途径及其固有的特殊性质 我们的线粒体细胞色素b5表达系统。的表达 细胞色素b5基因启动了血红素的合成，然后将其结合在一起。 在过表达的多肽中，从而简化了分离和 血红素的提纯。这种方法论不仅有利于研究 但它也将对其他感兴趣的研究人员有用 血红素蛋白质的核磁共振，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，以便与其重组其他蛋白质，或者 在模型化合物研究中使用它。二、阐明血红素的作用 细胞色素b5中的丙酸酯与生理伴侣结合 细胞色素c和P-450等蛋白质。为了达到这些目的，细胞色素b5 用13C标记的亚铁血红素将被用来提取结合等信息 位点、化学计量比和结合常数，这将对 了解电子转移反应。B5形成的络合物 与普遍存在的钙离子和镁离子有关，这两种离子最近被认为与 通过与暴露的血红素结合来调节b5的E1/2值 丙酸酯，也将用~(13)C和~(113)CD核磁共振波谱进行研究。 使用定点突变、核磁共振波谱和X射线 用结晶学和电化学技术研究主要因素 据信控制双组氨酸连接的细胞色素的还原潜力 B：这些因素是：a)水对疏水性血红素的可获得性 环境。B)带电残基之间的库仑相互作用接近 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N增量，导致更强或更弱 Fe-N键。D)组氨酰咪唑轴向平面的几何排列 这会受到轴向周围的氢键网络的影响 莱兰德。这些信息将有助于详细了解 双-组氨酸连接细胞色素b的结构功能，并致研究人员 对分子模型领域感兴趣的18，19，一类新的 参与氧化还原催化的金属蛋白的简化版本和在 能量转换。

项目成果

Heme oxygenase, steering dioxygen activation toward heme hydroxylation.

血红素加氧酶，将双氧激活转向血红素羟基化。

• DOI: 10.1016/j.jinorgbio.2004.09.016
• 发表时间: 2005
• 期刊: Journal of inorganic biochemistry
• 影响因子: 3.9
• 作者: Rivera,Mario;Zeng,Yuhong
• 通讯作者: Zeng,Yuhong

Mitochondrial and microsomal ferric b5 cytochromes exhibit divergent conformational plasticity in the context of a common fold.

线粒体和微粒体铁 b5 细胞色素在共同折叠的背景下表现出不同的构象可塑性。

• DOI: 10.1021/bi050564l
• 发表时间: 2005
• 期刊: Biochemistry.
• 作者: Simeonov,Mario;Altuve,Adriana;Massiah,MichaelA;Wang,An;Eastman,MargaretA;Benson,DavidR;Rivera,Mario
• 通讯作者: Rivera,Mario

Two distinct ferritin-like molecules in Pseudomonas aeruginosa: the product of the bfrA gene is a bacterial ferritin (FtnA) and not a bacterioferritin (Bfr).

• DOI: 10.1021/bi2004119
• 发表时间: 2011-06-14
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Yao H;Jepkorir G;Lovell S;Nama PV;Weeratunga S;Battaile KP;Rivera M
• 通讯作者: Rivera M

Biochemical and structural characterization of Pseudomonas aeruginosa Bfd and FPR: ferredoxin NADP+ reductase and not ferredoxin is the redox partner of heme oxygenase under iron-starvation conditions.

铜绿假单胞菌 Bfd 和 FPR 的生化和结构特征：铁氧还蛋白 NADP 还原酶而非铁氧还蛋白是缺铁条件下血红素加氧酶的氧化还原伴侣。

• DOI: 10.1021/bi7013135
• 发表时间: 2007
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Wang,An;Zeng,Yuhong;Han,Huijong;Weeratunga,Saroja;Morgan,BaileyN;Moënne-Loccoz,Pierre;Schönbrunn,Ernst;Rivera,Mario
• 通讯作者: Rivera,Mario

Conversion of mitochondrial cytochrome b5 into a species capable of performing the efficient coupled oxidation of heme.

将线粒体细胞色素 b5 转化为能够进行血红素有效偶联氧化的物质。

• DOI: 10.1021/bi9809324
• 发表时间: 1998
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Rodríguez,JC;Rivera,M
• 通讯作者: Rivera,M