DESIGN OF REDOX ACTIVE METAL HEME HYDRID ENZYMES

本提案的总体目标是通过设计，在氧化血红素酶的预存在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，以研究两个金属中心相互作用导致的电子转移、电子耦合和氧化还原反应。这种双核金属蛋白复合物可以作为天然存在的酶如锰依赖性木质素酶和细胞色素c氧化酶的机制和功能模型。在靠近血红素外围的细胞色素c过氧化物酶中引入金属结合位点，导致H2O2对Mn+2离子的酶依赖性氧化显著增强。进一步的研究将在两个截然不同的领域提供见解：1)通过比较这些杂化物的动力学、光谱学、能量学和协因子依赖性，可以对配体环境、金属-血红素偶联和氧化还原热力学等参数的相对重要性有重要的认识。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)在包含第二个氧化还原活性辅助因子的模型蛋白框架中，积累有关几个相关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VII中的实验旨在补充其他设计的金属蛋白的工作，但重点是金属辅助因子相互作用的细节。此外，我们将在项目项目中进行支持和合作研究，以帮助表征绿色荧光蛋白（GFP）发色团附近设计的金属位点。EPR、电化学、量热和荧光动力学实验旨在探索金属结合对发色团性质的影响，将为这些项目组成部分提供重要的反馈。通过这种方式，拟议的研究将通过为蛋白质结构中金属位点的设计和评估提供规则基础，从而直接影响项目的中心主题，以控制新型金属蛋白的调节，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.