HEMEPROTEINS: STATIC & RAPID KINETIC MAGNETIC STUDIES

血红素蛋白：静态

基本信息

批准号：
3337790
负责人：
John Philo
金额：
$ 13.73万
依托单位：
UNIVERSITY OF CONNECTICUT STORRS
依托单位国家：
美国
项目类别：
财政年份：
1979
资助国家：
美国
起止时间：
1979-07-01 至 1991-06-30
项目状态：
已结题

项目摘要

The techniques of magnetic susceptibility and rapid reaction kinetics will be used to study the structure, function and dynamics of the active sites of metalloproteins. To accomplish this we have developed a unique high-sensitivity time-resolved magnetic susceptometer. This instrument, which is based on recent advances in superconducting technology, is 100 times more sensitive than conventional susceptometers and provides microsecond time resolution for studying the rapid kinetics of changes in oxidation or spin state of the active site metal ions as the protein functions. A number of the proposed studies are directed at a detailed understanding of the mechanism of cooperative oxygen binding to hemoglobin. Such an understanding is relevant to both the physiology of O2 transport, and a general understanding of allosteric enzymes and protein control of metal ion activity. The dominant proposals for the hemoglobin cooperative mechanism invoke coupling between the spin state of the heme iron and changes in protein structure. Our recent magnetic studies of human, mutant, and carp ferric (met) hemoglobins suggest that this is incorrect. We propose further critical tests of the Perutz Stereochemical model of cooperativity, using Time Resolved Magnetic Susceptibility (TREMS) and optical kinetic studies of valence hybrid hemoglobins to further quantitate the linkage between spin states and protein structure. We also propose studies of the kinetics of changes in quaternary structure in hemoglobin, and tests of recent proposals that there are intermediate quaternary structures and that transitions between them are governed by the distribution as well as the number of O2 bound per hemoglobin tetramer. We are also proposing the first TREMS investigations of some enzymes involved in important oxidation-reduction reactions, and which contain several redox-active metal centers. It is hoped that these measurements will help to delineate the electronic structure of reaction intermediates, and possibly also the dynamics of electron flow between metal sites. Such information should contribute to a better understanding of how these enzymes are able to store redox energy from single electron transfers in order to carry out multi-electron oxidations or reductions. Other magnetic studies of heme, copper, and iron-sulfur proteins are proposed to answer specific questions about their electronic configurations and active site structures.

磁化率与快速反应技术动力学将用于研究结构、功能和动力学金属蛋白的活性位点。为了实现这一目标，我们开发了一种独特的高灵敏度时间分辨磁磁敏感计。该仪器是基于最近超导技术的进步，是其100倍比传统的感受器更灵敏，并提供用于研究快速动力学的微秒时间分辨率活性位点金属离子的氧化或自旋状态的变化为蛋白质的功能。一些拟议的研究针对详细的了解协同氧结合的机制血红蛋白。这种理解与双方都相关 O2 运输的生理学，以及一般理解变构酶和蛋白质控制金属离子活性。这血红蛋白合作机制的主导建议调用血红素铁的自旋状态和蛋白质结构的变化。我们最近的磁学研究人类、突变体和鲤鱼铁 (met) 血红蛋白表明这是不正确的。我们建议对佩鲁茨进行进一步的关键测试使用时间分辨的协同立体化学模型磁化率 (TREMS) 和光学动力学研究价杂合血红蛋白以进一步定量这种联系自旋态和蛋白质结构之间。我们还建议四级结构变化动力学研究血红蛋白，以及最近提出的测试中间四级结构和之间的过渡它们受到 O2 分布和数量的控制每个血红蛋白四聚体结合。我们还提议对一些国家进行首次 TREMS 调查参与重要氧化还原反应的酶，以及其中含有多个氧化还原活性金属中心。希望这些测量将有助于描绘电子反应中间体的结构，也可能是金属位点之间电子流的动力学。此类信息应该有助于更好地理解这些酶如何能够储存来自单电子转移的氧化还原能量从而进行多电子氧化或还原反应。血红素、铜和铁硫蛋白的其他磁性研究建议回答有关其电子设备的具体问题配置和活性位点结构。