MOSSBAUER STUDIES OF HEME MODELS AND MUTANT MYOGLOBINS

在研究铁蛋白的结构-功能关系时， 事实证明，EPR和穆斯堡尔等光谱技术对 了解活性中心的化学性质。《光谱》 参数提供了将一种铁蛋白与其他铁蛋白进行比较的清晰方法 具有不同的催化性能。拟议的项目涉及 血红素模型化合物和突变形式的物理表征 肌红蛋白。我们对具有a)阳离子的血红素模型化合物感兴趣 自由基团，b)铁的“混合自旋”铁状态，或c)低自旋铁 具有不同寻常的DXY基态。阳离子自由基是 许多哺乳动物血红素酶催化循环中的中间状态， 比如过氧化物酶。我们对血红素模型的研究将在#年完成。 与W.R.Scheidt博士合作。我们希望澄清一下 血红素的外周成分和轴向配基影响1) 铁的基态自旋态，2)阳离子占据的轨道 3)铁与自由基间的磁性耦合。我们 将专门使用穆斯堡尔谱和EPR谱来研究血红素 与八乙基卟啉(OEP)模型有关的化合物 [Fe(OEP‘)C_1]_2(SbC_(16))_2及其多一个电子的等价化合物 和一个反离子。其光谱和磁学参数 模型将通过计算机模拟确定，并将进行关联 具有来自X射线衍射测量的结构信息。此案 对于铁蛋白来说，首先令人信服的是它处于“混合自旋”状态 为铁细胞色素c‘制造的。从那时起，许多其他的酶和模型 化合物已经表现出这种状态。我们想要澄清一下化学物质 稳定这种状态的必要条件。低自旋的亚铁血红素 感兴趣的模型是与四苯基卟啉(TPP)相关的模型。 模型[Fe(TPP)(CNBu)2]，与几乎任何其他低自旋铁不同 系统，具有轴向低=自旋EPR谱，并可能具有未配对的 DXY轨道上的电子。我们希望阐明它们的成键性质 需要轴向配位体才能产生这种不寻常的状态。 肌红蛋白研究将与S·G·斯利格博士合作完成， 并将重点阐明铁配体的性质如何结合 位点被多肽主干的修饰所干扰。 重组技术将用于产生E和F的突变 锁链。突变的肌红蛋白将通过观察 CO和O2相对于野生型和穆斯堡尔谱的结合性质 光谱学和电子顺磁共振。穆斯堡尔参数将在一个 水晶场模型，它应该提供对水晶场如何 电势与蛋白质中微小的结构变化相关，以及如何 这些结构的变化反过来影响了血红素的反应性。 结合部位。为了方便分析穆斯堡尔的所有数据 在这些系统中，我们将开发一种迭代多参数计算机拟合 基于现代最小化算法的程序。

In studying structure-function relationships of iron proteins, spectroscopic techniques like EPR and Mossbauer have proved invaluable for understanding the chemical nature of the active center. The spectroscopic parameters provide a clear way of comparing one iron protein with others having different catalytic properties. The proposed project involves the physical characterization of heme model compounds and mutant forms of myoglobin. We are interested in heme model compounds having a) pi-cation radicals, b) a ferric "mixed-spin" iron state, or c) a low-spin ferric iron with an unusual dxy ground state. The cation radicals are models for intermediate states in the catalytic cycles of many mammalian heme enzymes, such as the peroxidases. Our studies on heme models will be done in collaboration with Dr. W. R. Scheidt. We hope to clarify how the peripheral constituents and axial ligands of the heme influence 1) the ground spin state of the iron, 2) the orbital occupied by the cation radical, and 3) the magnetic coupling between the iron and the radical. We will specifically use Mossbauer and EPR spectroscopy to study heme compounds related to the octaethyl porphyrin (OEP) model [Fe(OEP')C1]2(SbC16)2 and the equivalent compound having one more electron and a single counterion. The spectroscopic and magnetic parameters of the models will be determined by computer simulation, and will be correlated with structural information from x-ray diffraction measurements. The case for an iron protein being in the "mixed spin" state was first convincingly made for ferricytochrome c'. Since then, many other enzymes and model compounds have exhibited this state. We would like to clarify the chemical conditions necessary to stabilize this state. The low-spin ferric heme models of interest are those related to the tetraphenyl porphyrin (TPP) model [Fe(TPP)(CNBu)2]+ which, unlike nearly any other low-spin ferric system, has an axial low=spin EPR spectrum and may have its unpaired electron in a dxy orbital. We hope to elucidate the bonding properties required of the axial ligands in order to produce this unusual state. The myoglobin studies will be done in collaboration with Dr. S. G. Sligar, and will focus on clarifying how the properties of the iron ligand binding site are perturbed by modifications of the polypeptide backbone. Recombinant techniques will be used to generate mutations of the E and F chains. The mutant myoglobins will be investigated by looking at the binding properties of CO and O2 relative to the wild type, and by Mossbauer spectroscopy and EPR. The Mossbauer parameters will be analyzed in a crystal field model, which should afford insight into how crystal field potentials correlate with small structural changes in a protein, and how those structural changes in turn influence the reactivity of the heme binding site. To facilitate the analysis of the Mossbauer data on all of these systems, we will develop an iterative multiparameter computer fitting program based on modern minimization algorithms.