DESIGN OF REDOX ACTIVE METAL HEME HYDRID ENZYMES

这一建议的总体目标是通过设计在氧化血红素酶的预先存在的血红素辅助因子附近引入一系列氧化还原活性金属中心的结合位点，以研究由于两个金属中心相互作用而产生的电子转移、电子耦合和氧化还原反应。这种双核金属蛋白复合体可以作为自然产生的酶的机制和功能模型，如依赖锰的木质素酶和细胞色素C氧化酶。细胞色素c过氧化物酶在血红素外围引入金属结合部位，可显著增强过氧化氢对Mn2+的酶依赖性氧化作用。继续的研究将在两个截然不同的领域提供见解：1)通过比较这些杂化化合物的动力学、光谱、能量学和辅因子依赖性，可以获得对配体环境、金属-血红素偶联和氧化还原热力学等参数的相对重要性的重要见解。因此，这些研究可能为含有相互作用的血红素和金属中心的自然系统的功能提供尚未解决的问题的答案。2)在含有第二氧化还原活性辅因子的具有良好特征的模型蛋白质框架的背景下，积累关于几个相关设计的金属结合位点的结合特异性、热力学和结构参数的新信息，将为金属蛋白设计计划的总体设计目标提供重要的输入。项目七中的实验旨在补充其他设计的金属蛋白的工作，但重点放在金属-辅因子相互作用的细节上。此外，我们将在该计划项目中进行支持性和协作性研究，以帮助确定绿色荧光蛋白(GFP)发色团附近设计的金属位点的特征。旨在探索金属结合对发色团性质的影响的EPR、电化学、量热和荧光动力学实验将对这些项目组件提供重要的反馈。这样，拟议的研究将直接影响方案项目的中心主题，为设计和评估蛋白质结构中的金属位点提供规则基础，目的是控制新金属蛋白的调节、折叠和功能。

The overall goals of this proposal are to introduce, by design, a series of binding sites for redox active metal centers near the pre-exiting heme co-factor of an oxidative heme enzyme in order to study the electron-transfer, electronic coupling, and redox reactions that result from the interaction of the two metal centers. Such binuclear metalloprotein complexes may serve as mechanistic and functional models of naturally occurring enzymes such as manganese dependent ligninase and cytochrome c oxidase. Metal binding sites have been introduced into cytochrome c peroxidase near the heme periphery that result in significantly enhanced enzyme dependent oxidation of Mn+2 ions by H2O2. Continued studies will provide insights in two distinctly different arenas: 1) By comparing the kinetics, spectroscopy, energetics and co- factor dependence of these hybrids, significant insight may be obtain into the relative importance of parameters such as ligand environment, metal-heme coupling, and redox thermodynamics. Thus, these studies may provide answers to unresolved questions about the function of natural systems containing interacting heme and metal centers. 2) The accumulation of new information on the binding specificity, thermodynamics and structural parameters of several related designed metal binding sites in the context of a well characterized model protein framework containing a second redox active co-factor will provide important input for the overall design goals of the metalloprotein design program. The experiments in project VII are meant to complement work on other designed metalloproteins, yet focus upon details of metal- co-factor interactions. In addition, we will perform supporting and collaborative studies within the program project to help characterize designed metal sites near the chromophore of green fluorescent protein (GFP). EPR, electrochemistry, calorimetry and fluorescence kinetics experiments intended to explore the effects of metal binding on the properties of the chromophore will provide significant feedback on these project components. In this way, the proposed studies will directly impact on the central themes of the program project by contributing to a rule base for the design and evaluation of metal sites within protein structures for the purpose of controlling the regulation, folding and function of novel metalloproteins.