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MAGNETIZATION STUDIES OF METALLOPROTEINS

金属蛋白质的磁化研究

基本信息

批准号：
3281190
负责人：
EDMUND P DAY
金额：
$ 11.4万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-08-01 至 1991-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3281190
关键词：
Escherichia coli Mossbauer spectrometry bacterial proteins biomagnetism measurement cytochrome oxidase electron spin resonance spectroscopy heme magnetism metalloproteins oxidation reduction reaction oxidoreductase photolysis sulfite reductase superoxide dismutase

项目摘要

Precise magnetization data on small volume (less than 200 mu L), low concentration (more than 100 mu M) metalloprotein samples can now be obtained using a commercial SQUID susceptometer and techniques we have developed. This data can be properly interpreted only if paramagnetic impurities and sample heterogeneities are also measured using a variety of spectroscopic techniques - preferably applied to the magnetization sample itself. Using this approach it is possible to measure the spin, spin concentration, zero-field splitting parameters, and exchange coupling of paramagnetic centers in metalloproteins. We will study selected samples of 57Fe enriched cytochrome oxidase from T. thermophilus after characterization by Mossbauer spectroscopy. We will select for sample homogeneity and purity. Magnetization data of these selected samples will be used to measure the strength of the exchange coupling at the a3:Cua3 site. The active site of E. Coli sulfite reductase (and of spinach nitrite reductase) consists of a four-iron cluster coupled to a siroheme. This coupled site can accommodate two electrons. We will determine the spin of the singly reduced state of this enzyme. The siroheme in this singly reduced state may be in an unusual intermediate-spin ferrous state. We will study oxidized and reduced uteroferrin and its phosphate complexes to determine the exchange coupling of its two-iron center in each of these states. The magnetization and Mossbauer spectra of the phosphate complex of 5?Fe enriched reduced uteroferrin will be studied to understand the lack of EPR signal of this parmagnetic state. The number of c-type hemes in hydroxylamine oxidoreductase (HAO) is variously estimated between 7 and 11 per monomer. We will use the new magnetization techniques to measure the total heme concentration to plus/minus 5%. This, in turn, will be used to establish accurate extinction coefficients for preparations of this enzyme. We have developed a multi-instrument, boron nitride sample holder for combined study of 57Fe enriched magnetization samples with Mossbauer spectroscopy. We will extend this multi-instrument strategy beyond iron containing proteins by developing an optical cuvette for combined study of magnetization samples with low temperature

小体积（小于200 μ L）的精确磁化数据，低浓度（大于100 μ M）金属蛋白样品可以现在使用商业SQUID温度计获得，我们开发的技术。这些数据可以适当仅当顺磁性杂质和样品还使用各种光谱测量不均匀性技术-优选地应用于磁化样品本身。使用这种方法可以测量自旋，浓度、零场分裂参数和交换金属蛋白中顺磁中心的耦合。我们将研究选择的样品57铁富集细胞色素氧化酶来自里氏木穆斯堡尔谱表征后的嗜热菌谱我们将根据样品的均匀性和纯度进行选择。这些选定样品的磁化数据将用于测量在α 3：Cua 3位点的交换偶联强度。 E.大肠杆菌亚硫酸盐还原酶（和菠菜亚硝酸盐还原酶）由四个铁簇组成，西罗血红素。这个耦合位点可以容纳两个电子。我们将决定这种酶的单一还原态的自旋。在这种单一还原状态下的西罗血红素可能处于一种不寻常的中间自旋亚铁态。我们将研究氧化型和还原型子宫铁蛋白及其磷酸盐配合物，以确定其两个铁中心的交换耦合在每一个国家。磁化强度和穆斯堡尔谱 5的磷酸盐复合物？铁富集的还原型子宫铁蛋白将研究，以了解缺乏EPR信号的顺磁状态羟胺氧化还原酶（HAO）中C型血红素的数量在每个单体7和11之间进行了不同的估计。我们将使用新的磁化技术测定总血红素浓度为± 5%。这反过来将用于建立精确的消光系数，酵素我们开发了一种多仪器、氮化硼样品保持器用于57 Fe富集磁化样品与穆斯堡尔谱。我们将把这种多功能仪器通过开发一种光学的，低磁化强度样品联合研究用比色皿温度