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

生命科学化学项目资助北卡罗来纳州立大学的Reza Ghiladi教授进行题为“天然血红蛋白过氧化物酶和工程血红蛋白过氧化物酶的结构、光谱和机理研究”的研究。这项研究包括探索脱氢过氧化物酶（DHP）的机制和扩展底物谱，DHP是一种潜在的新型和新兴的双功能蛋白质，即血红蛋白过氧化物酶的原型。DHP是第一个确定具有生物学相关过氧化物酶活性的珠蛋白，因此提供了一个独特的系统来探索我们对金属蛋白机制，激活和设计的理解。尽管许多关于DHP的研究已经帮助阐明了它的一些机制途径，但人们仍然不清楚这种双功能蛋白是如何同时作为血红蛋白和过氧化物酶的。生化分析、结构研究和光谱研究（停流紫外可见、EPR、核磁共振、共振拉曼和Mössbauer）将用于解决与底物结合和从过氧化物酶活性铁和珠蛋白活性氧化亚铁状态激活有关的问题。由于酪氨酸自由基在dhp催化和h2o2依赖的三卤酚脱卤过程中介导电子转移，低场和高场EPR研究将直接用于确定蛋白质自由基形成的位置。DHP突变体在硝基酚污染物生物修复中的潜在应用也将被研究。金属蛋白在所有生物体中发挥着核心作用，然而金属蛋白相互作用的支配原理仍然是科学家们感兴趣的，他们正在寻求建立理解金属蛋白结构-功能关系的范例。综上所述，本提案中概述的研究目标利用了传统生物无机方法的优点，不仅可以提出和解决化学家感兴趣的关于生物系统和生命过程的各种问题，而且对化学以外的领域也可能具有重要意义。这些包括解决生物催化、酶机制和从头蛋白质工程的基本问题，从而允许生物修复、化学酶合成和可再生生物燃料生产的生物质转化等领域的进步。此外，由拟议研究计划与教育努力相结合所支持的教师-学者活动所产生的更广泛影响包括：i)继续通过“创造之星”外展倡议，为农村K-2和中学的科学教室提供关于生命过程化学的演示、讲座和课程计划；Ii)继续指导有才华的弱势民族高中生进行暑期研究实习，利用金属酶的结构-功能关系介绍基本的化学生物学概念；Iii)带领本科和研究生化学专业的学生进行跨学科的科学探究，利用分子生物学和光谱学的新兴技术，这些技术正在成为下一代生物无机化学家的可用技术；Iv)促进低收入或城市化地区以外的本科生进入多学科研究实验室，作为扩大代表性不足的少数民族参与的工具。该项目由分子和细胞生物科学部生物分子动力学、结构和功能组共同资助。

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.