Proton coupled electron transfer in ribonucleotide reductase

通过这一奖项，化学部门的生命过程化学项目资助佐治亚理工学院的Bridgette Barry教授研究蛋白质核糖核苷酸还原酶（RNR）中的质子偶联电子转移（PCET）反应。这种酶加速脱氧核糖核苷酸的产生，脱氧核糖核苷酸是DNA的组成部分，因此在细胞分裂中起着关键作用。脱氧核糖核苷酸是由核糖核苷酸通过带负电荷的电子远距离转移的过程形成的。电子的转移与带正电的质子的转移是协调的。Barry教授研究蛋白质运动或动力学如何促进和控制RNR中的质子耦合电子转移（PCET）反应。这个项目的智力价值在于产生对PCET反应的基本化学见解，这种反应在生物学中无处不在。巴里教授参与了斯佩尔曼学院（Spelman College）的联合教学和研究工作，斯佩尔曼学院是一所历史悠久的黑人女子学院或大学（HBCU）。此外，Barry博士正在与亚特兰大的视觉障碍者中心（Center for the visualimpaired）合作，开始为有视觉障碍的高中生和中学生提供STEM辅导。这一努力是佐治亚理工学院新生化学课程创新的基础，其目标是进一步和更广泛地提高视障人士对STEM学科的参与。本研究项目比较了从细菌和人类细胞中分离的1a类RNR酶的不同成员，目的是确定保守的PCET机制。这类酶由两种亚基组成，α和β。它们使用酪氨酸基（YO）。-微分辅因子位于β亚基中，作为α亚基中底物还原的自由基引发剂。该过程通过位于α和β亚基的酪氨酸残基的保守途径可逆的PCET发生。值得注意的是，自由基转移途径跨越35埃。巴里教授验证了一种假设，即自由基通过α / β界面转移会改变YO的构象。自由基引发剂，改变氢键成YO。并改变PCET通路上其他酪氨酸的构象和氢键。采用了反应诱导红外光谱（RIFT-IR）和紫外共振拉曼光谱（UVRR）两种技术。UVRR和RIFT-IR光谱通过同位素标记、溶剂同位素交换、位点定向诱变和使用非天然氨基酸的位点特异性诱变来分配。光谱结果通过比较密度泛函理论（DFT）对含酪氨酸肽的计算和酪氨酸自由基差异簇的模型来解释。酪氨酸自由基和酪氨酸单重态亚基的分子动力学模拟也用于模拟氧化还原连接的构象变化。该项目促进了对酶机制的基本理解，并有助于开发能量转化的仿生策略。该奖项反映了美国国家科学基金会的法定使命，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.