HEME PROTEIN STRUCTURE AND FUNCTION

在这份竞争性续签申请中提出的工作是 过渡金属蛋白及其络合物的继续研究 金属中心可以用各种物理技术来探测。 将特别注意博莱霉素(BLM)，一种 糖肽类抗生素，可切割DNA并需要转换 这项活动的金属。我们将确定金属配体在 Fe(III)-BLm和活化的BLm，阐明了 由磁场和频率决定的配位位置 电子自旋回波调制(ESEEM)研究。我们将决定 当药物与DNA结合时，结构是如何变化的。我们会 金属博莱菌素与齐聚物、低聚物相互作用的研究 多核苷酸作为关联所提出的机制的一种手段 DNA裂解具有结合药物的特异性。使用 将使用Fe(III)-、活化-和02Co(II)-BLM、ESEEM研究 确定顺磁探头到重离子的距离 特别标记在多核苷酸上的糖。共振拉曼 建议进行研究以阐明结合氧的结构。 一种激活的不切割DNA的贝洛霉素衍生物。AS 已经在机制上看到了根本的不同 铁-博莱霉素与纯化的DNA的作用与细胞核相比， 建议用实验来研究DNA切割活性。 并将其与体外机制联系起来。我们会 BLM抗菌作用中对铜的需要量评估 在金属硫蛋白水平升高而铜水平 被隔离了。我们将继续发展电子自旋 回波包络调制(ESEEM)光谱 注意170，23NA和39K相互作用与 Mn(II)-ATP中的激酶，既能量化相互作用的数量 并确定它们与顺磁的距离 中间。我们对取代咪唑的持续ESEEM研究- ~(14)N与铜(II)、血红素和铁(III)的相互作用 四苯基卟啉，铜氧化酶和线粒体的模型 细胞色素b，将评估立体AS的相对贡献。 相比于电子效应对电子-核耦合的影响。

The work proposed in this competing renewal application is to continue studies on transition metalloproteins and complexes whose metal centers can be probed by various physical techniques. Particular attention will be given to bleomycin (BLM), a glycopeptide antibiotic that cleaves DNA and requires a transition metal for this activity. We will determine that metal ligand in Fe (III)-BLM and activated BLM, elucidating the structure of the coordination site from magnetic field and frequency dependent electron spin echo modulation (ESEEM) studies. We will determine how the structure is change when the drug binds to DNA. We will study the interaction of metallo bleomycins with oligo- and polynucleotides as a means of relating the proposed mechanism of DNA cleavage to the specificity of binding of the drug. With Fe(III)-, activated- and 02Co(II)-BLM, ESEEM studies will be used to determine the distance of the paramagnetic probe to deuterons specifically labelled on polynucleotide sugar. Resonance Raman studies are proposed to elucidate the structure of bound oxygen in an activated belomycin derivative that does not cleave DNA. As fundamental differences have already been seen in the mechanism of Fe-bleomycin action with purified DNA as compare to cell nuclei, experiments are proposed to study the DNA cleavage activity in nuclei and to relate this to the in vitro mechanism. We will assess the requirement for copper in the antibiotic action of BLM in cells where metallothionein level are elevated and copper is sequestered. We will continue the development of electron spin echo envelope modulation (ESEEM) spectroscopy with particular attention to the study of 170, 23NA and 39K interactions with Mn(II)-ATP in kinase, both to quantify the number of interacting nuclei and to determine their distances from the paramagnetic center. Our continuing ESEEM studies with substituted imidazole- 14N interactions with Cu(II), heme, and Fe(III)- tetraphenylporphyrin, models for copper oxidase and mitochondrial cytochrome b, will assess the relative contributions of steric as compared to electronic effects on electron-nuclear coupling.