Photosystem II as a model protein for understanding metalloenzyme active site assembly

项目摘要 金属酶在整个生物学中普遍存在，并与多种人类疾病有关。我们之间的差距 对金属酶的理解在于它们的成熟度。生物发生中间产物先于生物形成 成熟的酶，必须严格调控，才能在活性部位正确组装金属辅酶因子。尽管 关于成熟金属酶及其生物发生，特别是活性部位组装的丰富知识还不是很好 明白了。光系统II(PSII)是一种光驱动的氧化还原酶，其活性部位含有金属因子 这是通过一系列称为光活化的逐步离子结合和光氧化事件来组装的。 光活化为理解金属辅因子组装提供了一个方便的模型，因为它是简单的；在 体外组装可以通过在溶液中简单地添加所需的离子来实现，并且组装提前了 提供光量子。此外，PSII结构研究的最新进展发展了一种 用于常规解决PSII组装中间体的高分辨率结构的平台。我们建议 表征PSII的生物发生中间体，可能揭示金属酶组装的普遍规律。 该项目的长期目标是为组装 PSII中的金属辅因子(训练阶段)，并揭示PSII生物发生中间体如何维持前体 对现役现场维护很重要的结构(独立阶段)。我们将使用结构性方法， 生化技术和计算建模，以实现以下三个具体目标：(1) 揭示PSII的放氧复合体是如何组装的，(2)揭示PSII的前体结构， 是组装其活性部位所必需的，以及(3)了解瞬时结合亚基在PSII中的作用 生物发生学。获得这样的洞察力可能有助于开发新的治疗方法和设计原则 全新的金属酶工程学。

Project Summary Metalloenzymes are ubiquitous throughout biology and implicated in a variety of human diseases. A gap in our understanding of metalloenzymes lies in their maturation. Biogenesis intermediates precede formation of the mature enzyme and must be tightly regulated to correctly assemble the metallocofactor in the active site. Despite the wealth of knowledge on mature metalloenzymes, their biogenesis, especially active site assembly, is not well understood. Photosystem II (PSII) is a light-driven oxidoreductase whose active site contains a metallocofactor that is assembled through a series of step-wise ion binding and photooxidation events called photoactivation. Photoactivation provides a convenient model for understanding metallocofactor assembly because it is facile; in vitro assembly may be achieved by simply adding the required ions in solution and assembly is advanced by providing light quanta. Furthermore, recent advances in structural investigation of PSII have developed a platform for routinely solving high-resolution structures of PSII assembly intermediates. We propose to characterize biogenesis intermediates of PSII that may reveal generalized rules for metalloenzyme assembly. The long-term goals of this project are to provide structural and functional bases for assembly of the metallocofactor in PSII (training phase), and reveal how PSII biogenesis intermediates maintain precursor structures important for active site maintenance (independent phase). We will use structural approaches, biochemical techniques, and computational modeling to achieve the following three specific objectives: (1) to reveal how the oxygen evolving complex of PSII is assembled, (2) to uncover precursor structures of PSII that are integral for assembling its active site, and (3) to understand the role of transiently-bound subunits during PSII biogenesis. Gaining such insight may allow for the development of new therapeutics and design principles for de novo metalloenzyme engineering.