Mechanistic and Structural Studies of Novel Heme-Containing Enzymes

智力上的功绩。含铁血红素过氧化物酶是一种催化有机底物氧化同时将过氧化氢还原为水的酶。将研究从海洋海虫中分离出的两种新型血红素过氧化物酶，一种能够将卤素离子(如氯和溴)结合到芳香分子中，另一种能够去除它们。卤代过氧化物酶，诺托马司氯过氧化物酶(NCPO)，卤代酚，不寻常的是需要有机黄素成分。两面体脱卤酶(DHP)使卤代酚脱卤化。令人惊讶的是，DHP的结构是珠蛋白--与血液中发现的蛋白质折叠相同--和肌红蛋白(Mb)相同。这是第一个已知的酶珠蛋白。这两种过氧化物酶的独特结构性质使人们怀疑它们是否利用了一种新的过氧化物酶机制。将遵循一项由假设驱动的四管齐下的实验计划。首先，快速动力学实验将探索DHP和NCPO催化的机理，并直接确定采用传统机理还是替代机理。其次，将广泛测试DHP和NCPO执行标准过氧化物酶反应的能力，以促进我们对DHP和NCPO功能的理解。第三，将准备设计的DHP和Mb突变体，以探索DHP血红素附近特定氨基酸的机制作用，并探索在血红素/珠蛋白环境中相对于Mb增强DHP功能的因素。第四，对NCPO进行克隆、表达和结构鉴定。得到了较小的NCPO晶体。NCPO是一种复杂的多亚基血黄素酶，其亚基的作用尚不清楚。这类具有过氧化物酶活性的酶尚未见报道。对其结构和机制的研究将为高等生物中的卤化作用提供新的信息。对复杂的多亚基系统的研究，如本文提出的那些，处于机械酶学的前沿。总体而言，这两种新的过氧化物酶挑战了目前关于过氧化物酶如何发挥作用的观点。这项研究将探索大自然如何将小珠蛋白折叠蛋白重新设计为过氧化物酶，从而扩大我们对此类酶结构/功能关系的理解范围。更广泛的影响。这项研究结合了化学、分子遗传学和进化论、结构生物学和光谱学的不同学科，并通过三个研究小组的定期会议促进了化学和生物学教职员工和学生之间的互动。从高中到研究生的学生都将接受培训。本科生将继续为研究做出重大贡献。这三名PI有良好的记录，最近培训了代表不足的少数族裔，包括两名非裔美国学生。其中一名PI主管SCienceLab，这是一个针对初中/高中科学教师和学生的外展项目。以生物脱氢过氧化物酶为中心设计的SCiLab将提供为期一天的实际操作、基于探究的实验室体验，并传达生物、化学和环境研究的相互联系的兴奋。本课程的目标是向学生展示如何将生物催化剂用于生物修复技术。此外，研究成果将继续用于教授研究生的酶学、结晶学和金属生物化学课程。所取得的基本化学/生物化学进展可能导致生物技术和生物修复等更广泛领域的应用的发展，并将对相关的生态和环境研究产生影响。

Intellectual Merit. Heme-iron containing peroxidases are enzymes that catalyze organic substrate oxidation while reducing hydrogen peroxide to water. Two novel heme peroxidases isolated from marine sea worms, one able to incorporate halide ions such as chloride and bromide into aromatic molecules and the other able to remove them, will be studied. The haloperoxidase, Notomastus lobatus chloroperoxidase (NCPO), halogenates phenols and is unusual in requiring an organic flavin component. Amphitrite ornata dehaloperoxidase (DHP) dehalogenates halophenols. Surprisingly, the structure of DHP is that of a globin - the same protein fold found in hemo- and myoglobin (Mb). It is the first known enzymatic globin. The unique structural properties of these two peroxidases bring into question whether they utilize a new peroxidase mechanism. A four-prong hypothesis-driven experimental plan will be followed. First, rapid kinetics experiments will probe the mechanism of DHP and NCPO catalysis and directly determine whether the traditional or an alternative mechanism is employed. Second, the ability of DHP and NCPO to carry out standard peroxidase reactions will be extensively tested in order to advance our understanding of DHP and NCPO function. Third, designed DHP and Mb mutants will be prepared to probe the mechanistic role of specific amino acids near the DHP heme and explore factors in the heme/globin environment that enhance DHP function relative to Mb. Fourth, NCPO will be cloned, expressed and structurally characterized. Small NCPO crystals have been obtained. NCPO is a complex multi-subunit hemoflavoenzyme; the roles of its subunits are unknown. Such enzymes with peroxidase activity have not yet been reported. Studies of its structure and mechanism will provide new information about halogenation in higher organisms. Investigations of complex multi-subunit-containing systems, such as those proposed herein, are at the cutting edge of mechanistic enzymology. Overall, the two novel peroxidases challenge the current view of how peroxidases function. The research will probe how nature has redesigned small globin-fold proteins into peroxidases and thereby will extend the boundaries of our understanding of structure/function relations in such enzymes. Broader Impacts. The research combines the diverse disciplines of chemistry, molecular genetics and evolution, structural biology, and spectroscopy, and promotes interaction among chemistry and biology faculty and students via regular meetings of the three research groups. Students, from high school to post-graduate, will be trained. Undergraduates will continue to make significant research contributions. The three PIs have strong records training underrepresented minorities recently including two African-American students. One of the PIs directs SCienceLab, an outreach program for middle/high school science teachers and students. A specific 'Enzyme-Catalyzed Toxic Cleanup' SCiLab designed around biological dehaloperoxidases will provide a daylong hands-on, inquiry-based lab experience and convey the excitement of interfacing biological, chemical and environmental research. The goal is to show students how biological catalysts might be used in bioremediation technology. Moreover, the research results will continue to be used teaching graduate enzymology, crystallography and metallobiochemistry courses. The fundamental chemical/biochemical progress achieved may lead to the development of applications in the broader areas of biotechnology and bioremediation and will impact related ecological and environmental research.