CAREER:Structural, Spectroscopic, and Mechanistic Investigations of Native and Engineered Hemoglobin-Peroxidases

生命科学化学计划资助北卡罗来纳州立大学的雷扎·吉拉迪教授获得职业奖项，以开展题为“天然和工程血红蛋白过氧化物酶的结构、光谱和力学研究”的研究。这项研究涉及探索脱氢过氧化物酶(DHP)的机制和扩大底物图谱，DHP是一种潜在的新的和新兴的双功能蛋白的原型，称为血红蛋白-过氧化物酶。DHP是第一个被发现具有生物相关过氧化物酶活性的珠蛋白，因此为我们探索金属蛋白的机制、激活和设计提供了一个独特的系统。尽管许多关于DHP的研究已经帮助阐明了它的几个机制，但人们仍然不知道这种双功能蛋白如何既作为血红蛋白又作为过氧化物酶。生化分析、结构研究和光谱研究(停流UV-可见、EPR、核磁共振、共振拉曼和穆斯堡尔)将用于解决与底物结合和激活过氧化物酶活性的铁和珠蛋白活性的含氧亚铁状态有关的问题。由于酪氨酸基在DHP催化和H_2O_2依赖的三卤酚的脱卤化反应中参与了电子转移，因此低场和高场EPR研究将被用于确定蛋白质自由基形成的位置。DHP的突变体也将被研究在硝基苯酚污染物的生物修复中的潜在应用。金属蛋白在所有生物体中发挥着核心作用，然而，金属-蛋白质相互作用的主导原理仍然引起科学家的兴趣，他们正在寻求建立理解金属蛋白结构-功能关系的范例。综合起来，这项建议中概述的研究目标利用了传统生物无机方法的优点，这种方法不仅能够提出和解决化学家感兴趣的关于生物系统和生命过程的问题类型，而且可能对化学以外的领域具有潜在的重要性。这些措施包括解决生物催化、酶机制和从头蛋白质工程的基本问题，从而允许在生物修复、化学酶合成和生物质转化用于可再生生物燃料生产等领域取得进展。此外，将拟议的研究计划与教育工作相结合所支持的教师-学者活动产生的更广泛的影响包括：i)继续通过“创星”外展计划为农村K-2和中学科学课堂提供关于生命过程化学的示范、讲座和教案；ii)继续指导有才华的、处于不利地位的少数族裔高中生进行为期一年的暑期研究实习，利用金属酶的结构-功能关系介绍基本的化学生物学概念；3)引导本科生和研究生进行跨学科的科学研究，利用分子生物学和光谱学的新兴技术，向下一代生物无机化学家提供这些新技术；4)促进来自低收入或城市化以外地区的本科生进入多学科研究实验室，以此作为扩大少数群体参与的工具。这个项目得到了分子和细胞生物科学部生物分子动力学、结构和功能集群的共同资助。

The Chemistry of Life Sciences Program funds Professor Reza Ghiladi of North Carolina State University for a CAREER award to carry out research entitled "Structural, Spectroscopic, and Mechanistic Investigations of Native and Engineered Hemoglobin-Peroxidases." This research involves probing the mechanism and expanding the substrate profile of dehaloperoxidase (DHP), the archetype of a potentially new and emerging class of bifunctional proteins known as the hemoglobin-peroxidases. DHP is the first globin identified to possess a biologically relevant peroxidase activity, and thus provides a unique system to explore our understanding of metalloprotein mechanism, activation, and design. Despite a number of studies on DHP that have helped to elucidate several of its mechanistic pathways, it is still not understood how this bi-functional protein can act as both a hemoglobin and a peroxidase. Biochemical assays, structural studies, and spectroscopic investigations (stopped-flow UV-visible, EPR, NMR, resonance Raman, and Mössbauer) will be used to address questions pertaining to substrate binding and activation from both the peroxidase-active ferric and globin-active oxyferrous states. As tyrosyl radicals are implicated in mediating electron transfer in the DHP-catalyzed and H2O2-dependent dehalogenation of trihalophenols, low- and high-field EPR studies will be directed towards identifying the location of protein radical formation. Mutants of DHP will also be investigated for potential application in bioremediation against nitrophenol pollutants. Metalloproteins play a central role in all living organisms, yet the governing principles of the metal-protein interaction continue to be of interest to scientists who are looking toward building paradigms for understanding metalloprotein structure-function relationships. When taken together, the research goals outlined within this proposal take advantage of the merits of a traditional bioinorganic approach that not only enable posing and addressing the types of questions about biological systems and life processes that are of interest to chemists, but can also potentially be of importance to areas outside of chemistry. These include addressing the fundamental issues of biocatalysis, enzyme mechanism, and de novo protein engineering, thus allowing for the advancement of areas as diverse as bioremediation, chemoenzymatic synthesis, and biomass conversion for renewable biofuel production. Additionally, the broader impacts resulting from the teacher-scholar activities supported by the integration of the proposed research plan with educational endeavors include the following: i) continue to provide rural K-2 and middle school science classrooms with demonstrations, lectures, and lessons plans for teachers on the chemistry of life processes through the "Creating STARS" outreach initiative; ii) continue the mentoring of talented, disadvantaged minority high school students for summer long research internships, using the structure-function relationship in metalloenzymes to introduce basic chemical biology concepts; iii) lead undergraduate and graduate chemistry students in interdisciplinary scientific inquiry that utilizes emerging techniques in molecular biology and spectroscopy that are becoming available to the next generation of bioinorganic chemists; iv) promote the placement of undergraduate students from low-income or outside-urbanization locations into multidisciplinary research labs as vehicles for broadening the participation of underrepresented minorities. This project is receiving co-funding from the Biomolecular Dynamics, Structure and Function Cluster in the Division of Molecular and Cellular Biosciences.