Proton coupled electron transfer in ribonucleotide reductase

有了这个奖项，在化学部生命过程计划的化学资助教授布里奇特巴里在技术的格鲁吉亚研究所研究质子耦合电子转移（PCET）反应的蛋白质，核糖核苷酸还原酶（RNR）。 这种酶加速脱氧核糖核苷酸的产生，脱氧核糖核苷酸是DNA的构建块，因此在细胞分裂中起着关键作用。脱氧核糖核苷酸由核糖核苷酸通过涉及带负电荷的电子长距离转移的过程形成。 电子的转移与带正电荷的质子的转移相协调。 巴里教授研究蛋白质运动或动力学如何促进和控制RNR中的这些质子耦合电子转移（PCET）反应。 该项目的智力价值在于对PCET反应产生基本的化学见解，这在生物学中无处不在。教授巴里参加了联合教学和研究工作，与教授丽莎希巴德在斯佩尔曼学院，历史上黑人学院或大学（HBCU）的妇女合作。此外，巴里博士正在发起与亚特兰大视障中心的合作，开始对有视力障碍的高中和初中学生进行STEM辅导。这项工作是格鲁吉亚理工学院大一化学课程创新的基础。该研究项目比较了从细菌和人类细胞中分离出的1a类RNR酶的不同成员，目的是定义保守的PCET机制。 这类酶由两种类型的亚基组成，α和β。 它们使用酪氨酰基自由基（YO.）-位于β亚基中的二铁辅因子作为自由基引发剂用于α亚基中的底物还原。 该过程通过位于α和β亚基中的酪氨酸残基的保守途径上的可逆PCET发生。 值得注意的是，自由基转移途径跨越35埃。 巴里教授检验了这样一个假设，即穿过α/β界面的自由基转移改变了YO的构象。自由基引发剂，改变与YO的氢键。和二价铁簇，并改变PCET途径上其他酪氨酸的构象和氢键。 两种技术，即反应诱导傅立叶变换红外光谱（RIFT-IR）和紫外共振拉曼光谱（UVRR），被采用。UVRR和RIFT-IR光谱通过同位素标记、溶剂同位素交换、定点诱变和使用非天然氨基酸的位点特异性诱变进行归属。 光谱结果进行解释，通过比较，密度泛函理论（DFT）计算的酪氨酸含肽和模型的酪氨酰自由基differic集群。 分子动力学模拟的亚基在酪氨酰自由基和酪氨酸单线态也被用来模拟氧化还原连接的构象变化。该项目推进了对酶机制的基本理解，并有助于开发用于能量转换的仿生策略。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Professor Bridgette Barry at the Georgia Institute of Technology to investigate proton coupled electron transfer (PCET) reactions in the protein, ribonucleotide reductase (RNR). This enzyme speeds up the production of deoxyribonucleotides, which are building blocks of DNA, and thus plays a pivotal role in cell division. The deoxyribonucleotides are formed from ribonucleotides by a process that involves transfer of negatively-charged electrons over a long distance. The transfer of the electrons is coordinated with the transfer of positively-charged protons. Professor Barry studies how protein motions or dynamics facilitate and control these proton-coupled electron transfer (PCET) reactions in RNR. The intellectual merit of this project lies in the generation of fundamental chemical insights into PCET reactions, which are ubiquitous in biology. Professor Barry participates in a joint teaching and research effort in collaboration with Professor Lisa Hibbard at Spelman College, an Historically Black College or University (HBCU) for women. In addition, Dr. Barry is initiating a collaboration with the Center for the Visually Impaired in Atlanta to begin STEM tutoring of high school and middle school students with vision impairments. This effort serves as the basis for innovation in the freshman chemistry curriculum at Georgia Tech. The goal is to further and more broadly enhance the participation of visually impaired individuals in STEM disciplines.This research project compares different members of the Class 1a RNR enzymes isolated from bacteria and human cells, with the goal of defining conserved PCET mechanism(s). Enzymes from this class are composed of two types of subunits, alpha and beta. They employ a tyrosyl radical (YO.)-diferric cofactor situated in the beta subunit as a radical initiator for substrate reduction in the alpha subunit. This process occurs through reversible PCET over a conserved pathway of tyrosine residues situated in the alpha and beta subunits. Remarkably, the radical transfer pathway spans 35 angstroms. Professor Barry tests the hypothesis that radical transfer across the alpha/beta interface changes the conformation of the YO. radical initiator, alters hydrogen bonding to YO. and the diferric cluster, and changes the conformation and hydrogen bonding of other tyrosines on the PCET pathway. Two techniques, namely reaction-induced FT-IR (RIFT-IR) and UV resonance Raman (UVRR) spectroscopy,, are employed. The UVRR and RIFT-IR spectra are assigned by isotopic labeling, solvent isotope exchange, site-directed mutagenesis, and site-specific mutagenesis using unnatural amino acids. Spectral results are interpreted by comparison to Density Functional Theory (DFT) calculations on tyrosine-containing peptides and on models of the tyrosyl radical-differic cluster. Molecular dynamics simulations of the subunit in the tyrosyl radical and tyrosine singlet states are also used to simulate redox-linked conformational changes. This project advances the fundamental understanding of enzyme mechanism and aids in the development of biomimetic strategies for energy conversion.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.