13C LABELED GLYCEROL

SIR提供D，L-[咪唑-N2]组氨酸; Ig 甲苯-4-单加氧酶（T4 MO）催化NADH-和O2-依赖性的 甲苯，吲哚，萘，氯仿， 三氯乙烯和其它烃类。 酶复合物由 由四种组分组成：12.5kDa的Rieske铁氧还蛋白（T4 MOC， 本研究的主题）一个11.6 kDa的调节蛋白（T4 MOD）;一个216 kDa二铁羟化酶（T4 MOH）;和36 kDa NADH氧化还原酶。 我们 正在使用多核高场核磁共振研究T4 MOC。 我们的计划是 广泛利用为研究其他问题而开发的方法， 铁硫蛋白在国家磁共振设施， 麦迪逊。 我们对T4 MOC的研究代表了第一个详细的NMR 一种Rieske蛋白的研究 因此，他们将提供一个 评价和改进现有NMR的实验框架 方法，并将提供电子和动态信息， 氧化还原状态、质子交换和自旋之间的相关性 离域作用可能有助于Rieske之间的电子转移 和DFIRON中心。 本报告将涉及两个不同的领域 研究工作：1）超精细位移共振的性质 与T4 MOC的氧化和还原状态相关;和2）2-D 和3-D NMR研究的抗磁共振所产生的 T4 MOC和T4 MOD的蛋白质骨架。 本研究的目的 是了解蛋白质的分子特性（T4 MOD）， 二铁羟化酶（T4 MOH），导致100至300倍 催化速率的提高。 T4 MO系统提供独特的 有机会研究Rieske之间电子转移的能量学 和二铁中心，并研究基于蛋白质的氧合酶控制 调节蛋白如T4 MOD的催化作用， 实验系统 因此，很可能有意义的新见解 关于热力学和生物控制的二铁加氧酶 将从拟议的活动中获得。

The SIR provided D, L-[imidazole-"N2]Histidine; lg Toluene-4-monooxygenase (T4MO) catalyzes the NADH- and 02-dependent oxidation of toluenen, indole, naphthalene, chloroform, trichlorethylene, an dother hydrocarbons. The enzyme complex consists of four components: a 12.5 kDa Rieske ferredoxin (T4MOC, the primary subject of this study) an 11.6 kDa regulatory protein (T4MOD); a 216 kDa diiron hydroxylase (T4MOH); and a 36kDa NADH oxidoreductase. We are studying T4MOC using multinuclear, high field NMR. Our plan is to make extensive use of methods developed for the study of other iron-sulfur proteins at the National Magnetic Resonance Facility at Madison. Our studies of T4MOC represent the first detailed NMR studies of a Rieske protein. As such, they will provide an experimental framework for evaluating and improving existing NMR methods, and will provide eletronic and dynamic information about correlations between redox states, proton exchange, and spin delocalization that may contribut to electron transfer between Rieske and dfiron centers. Two distinct areas will be addressed in this research effort: 1)properties of hyperfine-shifted resonances associated with the oxidized and reduced states of T4MOC; and 2) 2-D and 3-D NMR studies of the diamagnetic resonances arising from the protein backbones of T4MOC and T4MOD. The objective of this research is to understand the molecular properties of protein (T4MOD), and the diiron hydroxylase (T4MOH) that lead to the 100- to 300-fold enhancements in catalytic rate. The T4MO system offers a unique opportunity to study the energetics of electron transer between Rieske and diiron centers, and to study the protein-based control of oxygense catalysis by regulatory proteins such as T4MOD within a single experimental system. Thus, it is likely that significan new insights about thermodynamic and biological control of diiron oxygenase enzymes will be obtained from the proposed activity.