HEME PROTEIN STRUCTURE AND FUNCTION

本竞争性更新申请中提出的工作是 继续研究过渡金属蛋白和复合物， 可以通过各种物理技术探测金属中心。 将特别注意博莱霉素（BLM）， 一种糖肽抗生素，可切割DNA，需要一种过渡 金属对于这项活动。 我们将确定金属配体， Fe（III）-BLM和活化的BLM，阐明了 磁场和频率依赖的配位位点 电子自旋回波调制（ESEEM）研究。 我们将确定 当药物与DNA结合时结构是如何变化的。 我们将 研究金属博莱霉素与寡聚和 多核苷酸作为一种手段，涉及提出的机制， DNA裂解能特异性结合药物。 与 将使用Fe（III）-、活化-和02 Co（II）-BLM、ESEEM研究 以确定顺磁探针到氘核的距离 特异性标记在多核苷酸糖上。 共振拉曼 研究提出，以阐明结构的束缚氧在 一种不裂解脱氧核糖核酸的活性贝洛霉素衍生物。 作为 基本的差异已经在机制中看到， 与细胞核相比，铁-博来霉素对纯化DNA的作用， 实验提出了研究DNA切割活性， 核，并将其与体外机制联系起来。 我们将 评估BLM抗菌作用中铜的需求 在金属硫蛋白水平升高且铜 与世隔绝 我们将继续发展电子自旋 回波包络调制（ESEEM）光谱学， 注意研究170、23 NA和39 K与 Mn（II）-ATP在激酶中，两者都定量相互作用的数量。 并确定它们与顺磁核的距离 中心 我们对取代咪唑的持续ESEEM研究- 14 N与Cu（II）、血红素和Fe（III）的相互作用- 四苯基卟啉，铜氧化酶和线粒体模型， 细胞色素B，将评估空间的相对贡献， 与电子核耦合的电子效应相比。

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.