MAGNETIZATION STUDIES OF METALLOPROTEINS

小体积上的精准磁化数据(L 200亩以下)， 低浓度(超过100亩M)的金属蛋白样品可以 现在使用商业鱿鱼感受器和 我们开发的技术。该数据可以正确地 仅在顺磁性杂质和样品 异质性也可以用各种光谱来测量。 技术--最好应用于磁化样品本身。 使用这种方法可以测量自旋、自旋 浓度、零场分裂参数和交换 金属蛋白中顺磁中心的偶联。 我们将研究精选的57Fe富铁细胞色素氧化酶样品 穆斯堡尔定征后的嗜热链霉菌 光谱学。我们将根据样品的均质性和纯度进行选择。 这些选定样品的磁化数据将用于 测量A3：CuA3部位的交换耦合强度。 大肠杆菌亚硫酸盐还原酶(和菠菜)的活性部位 亚硝酸盐还原酶)由一个四铁簇连接到一个 西罗西米亚人。这个耦合位置可以容纳两个电子。我们 将决定这种酶的单一还原状态的自旋。 处于这种单一还原状态的西罗西米亚人可能处于一种不寻常的状态 中间自旋的亚铁态。 我们将研究氧化和还原的子宫铁蛋白及其磷酸盐 确定其两铁中心的交换耦合的络合物 在这些州中的每一个。磁化强度和穆斯堡尔谱 富含5？Fe的还原子宫铁蛋白的磷酸盐络合物 被研究以理解这个抛物线的EPR信号缺乏 州政府。 羟胺氧化还原酶(HAO)中C型血红素的数量 根据不同的估计，每个单体在7到11之间。我们将使用 测定总铁血红素的磁化新技术 浓度为正负5%。这反过来将被用来 为该制剂建立准确的消光系数 酵素。 我们研制了一种多功能仪器--氮化硼样品架。 用于57Fe富磁化样品的联合研究 穆斯堡尔谱学。我们将推广这一多功能仪器 超越含铁蛋白质的战略通过开发一种光学 用于低磁化样品联合研究的试管 温度

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