CAREER: Structure And Function Of Sulfite Reductase Teach About Fundamental Biology

智力上的优点硫的减少是生命的核心，作为一个残留的提醒地球之前出现的氧为基础的能源货币。硫化物形式的有机硫仍然是所有生物体的必需营养素，但高等真核生物无法还原无机硫（IV），因此依赖细菌，古生菌，酵母和植物来生产可用的硫。亚硫酸盐还原为硫化物的六电子还原是生物-地质硫循环的核心，并且由单一酶-亚硫酸盐还原酶（SiR）催化。这种“高容量”的电子转移是硫和氮循环所特有的，代表了一个强有力的生化反应的例子，其中自然界比人类化学工程师更多才多艺。在一起，SiR在营养循环中的中心地位及其独特的生物化学特性，使研究SiR的机制和结构成为了解自然环境的重要基础。本项目的目标是通过实验研究SiR氧化还原酶还原硫过程中电子转移的化学和生物学，并通过实验室综合教育推广扩大这项研究的影响，教室实验结果将通过将X射线晶体学和单粒子低温电子显微镜（cryo-EM）的多分辨率信息与生化分析相结合，提高对硫还原机制的认识。具体而言，正在调查两个领域。首先，特定氨基酸在SiR活性中的作用将通过诱变、SiR活性测定和X射线晶体结构分析来探测。其次，将探索SiR亚基组装，多角度光散射，cryo-EM成像和单颗粒3DEM分析，以了解驱动硫还原的蛋白质间化学。更广泛的影响应用结构技术来回答有关环境细菌学的基本问题，通过提供对调节硫的微生物循环机制的深入了解，使整个社会受益。此外，探索硫还原途径为整合研究和教学提供了独特的平台。拟议的外联部分利用基于假设驱动的研究的多方面课堂练习，吸引各种学生参与。具体来说，从高中到博士后研究人员的学生将有针对性地加入实验室，在那里他们将接触到结构生物学的研究。佛罗里达州立大学拥有全球约20台Titan Krios低温透射电子显微镜，对这种独特仪器的培训是拟议研究的组成部分。此外，应用结构技术依赖于计算，数学，化学，生物化学和微生物学的各种工具，提供了一系列潜在的研究项目，共同关注回答生物学问题。与此同时，专门为佛罗里达州立大学第一年生物学导论课程开发的主动学习模块将加强和提高生物物理学、生物化学、分子生物学和细胞生物学的课堂课程。SiR是课堂练习的一个吸引人的，有效的基础，因为它是一个新的模型来解释大多数讲座课程或教科书中没有探索的基本生命科学概念。这些课堂模块与有志于教授K-12科学和技术的学生一起开发，进一步扩大了这一提案对改善美国普通科学教育的长期影响。

Intellectual MeritSulfur reduction is central to life as a vestigial reminder of Earth before the emergence of an oxygen-based energy currency. Organic sulfur in the form of sulfide remains an essential nutrient in all organisms, but higher eukaryotes are unable to reduce inorganic sulfur(IV) so rely on bacteria, archaea, yeast, and plants to produce useable sulfur. The six-electron reduction of sulfite to sulfide is central to the bio-geo sulfur cycle and is catalyzed by a single enzyme, sulfite reductase (SiR). This "high-volume" electron transfer is unique to sulfur and nitrogen cycles and represents one powerful example of a biochemical reaction where nature is more versatile than human chemical engineers. Together, the centrality of SiR to nutrient cycles and its unique biochemistry make a compelling case that research into its mechanism and structure lays important groundwork for understanding our natural environment.The goals of this project are to experimentally dissect the chemistry and biology of electron transfer in sulfur reduction by the oxidoreductase SiR and to broaden the impact of this research through integrated educational outreach in the laboratory and classroom. Experimental results will advance knowledge about mechanisms of sulfur reduction by combining information at multiple resolutions from x-ray crystallography and single particle cryogenic electron microscopy (cryo-EM) with biochemical analysis. Specifically, two areas are under investigation. First, the role of specific amino acids in SiR activity will be probed with mutagenesis, SiR activity assays, and x-ray crystallographic structural analysis. Second, the SiR subunit assembly will be explored, with multi-angled light scattering, cryo-EM imaging, and single particle 3DEM analysis, to understand inter-protein chemistry that drives sulfur reduction,. Broader ImpactsApplying structural techniques to answer fundamental questions about environmental bacteriology benefits society at large by providing insight into the mechanisms that regulate the biogeological cycling of sulfur. In addition, exploring sulfur reduction pathways provides a unique platform for integrating research and teaching. The proposed outreach component uses multifaceted classroom exercises based on hypothesis-driven research to engage a range of students. Specifically, students from high school to postdoctoral researchers will be targeted to join the laboratory where they will be exposed to research in structural biology. Florida State University is home to one of approximately 20 Titan Krios cryogenic transmission electron microscopes worldwide and training on this unique instrument is integral to the proposed research. Further, the applied structural techniques rely on diverse tools from computation, mathematics, chemistry, biochemistry, and microbiology, providing a range of potential research projects with a common focus on answering biological questions. At the same time, active-learning modules developed specifically for a first year Introduction to Biology class at Florida State University will reinforce and enhance classroom lessons on biophysics, biochemistry, molecular biology and cellular biology. SiR is an engaging, effective foundation for classroom exercises because it is a novel model to explain basic life science concepts not explored in most lecture courses or textbooks. These classroom modules developed with, and for, students aspiring to teach K-12 science and technology further broadens the long-term impact of this proposal on improving general science education in the United States.