X-ray spectroscopy to characterize intermediates in the catalytic cycle of Mn-Fe and Fe-Fe ribonucleotide reductases

铁-氧超家族的酶的特征在于由原型的同源铁-铁辅因子，其作为O2活化催化剂起作用，涉及电子转移（ET）过程中的高价中间体。核糖核苷酸还原酶（RNR），在所有生物体中的DNA合成所必需的，和配体结合氧化酶，重要的挑战性的转换反应，是突出的例子。该领域的最新结果表明，在人类病原体沙眼衣原体（Ct）和结核分枝杆菌（Mt）的R2-同源亚基中，异源MnFe物种形成天然催化剂，而不是标准的FeFe辅因子，MnMn位点也是活性的。此外，这些辅因子可以在存在和不存在标准RNR的众所周知的酪氨酰基自由基的情况下起作用。这就要求对这种类型的双金属辅因子的机械概念进行全面的重新评估。新的晶体结构代表了重大进展，但只含有低价态的辅因子。我们的目的是解开的FeFe，MnFe，MnMn辅因子在其O2激活的高价态（III 2; III，IV; IV 2）在Ct R2和MT氧化酶蛋白质的分子结构和电子构型，使用先进的X射线吸收和发射光谱技术结合密度泛函理论计算（XAES-DFT）。这些参数与辅因子组装途径、金属位点质子化、蛋白质亚基之间的相互作用以及ET的启动和门控有关。我们从光谱学和理论中获得的新信息应该允许分配位点选择性金属掺入的先决条件，比较RNR和氧化酶中的结构-功能关系，揭示辅因子种类在O2活化和ET反应中的作用，改进高价蛋白质状态的晶体学策略，并构建详细的模型结构，以更深入地了解这种迷人的多种双金属辅因子的机制。

The iron-oxygen superfamily of enzymes is characterized by a prototypic homo-bimetallic FeFe cofactor, which functions as an O2-activating catalyst, involving high-valent intermediates in electron transfer (ET) processes. Ribonucleotide reductases (RNRs), essential for DNA synthesis in all organisms, and ligand-binding oxidases, important for challenging conversion reactions, are prominent examples. Recent results in the field suggest that in the R2-homologue subunits of the human pathogens Chlamydia trachomatis (Ct) and Mycobacterium tuberculosis (Mt) a hetero-bimetallic MnFe species forms the native catalyst instead of the standard FeFe cofactor and MnMn sites are also active. In addition, these cofactors can function in the presence and absence of the well-known tyrosyl radical of standard RNRs. This calls for a general re-evaluation of the mechanistic concept of this type of dimetal cofactor. New crystal structures represent significant progress, but contain only low valence states of the cofactors. We aim at unravelling the molecular structure and electronic configuration of the FeFe, MnFe, and MnMn cofactors in their O2-activated high-valent states (III2; III,IV; IV2) in Ct R2 and Mt oxidase proteins using advanced X-ray absorption and emission spectroscopy techniques in combination with density functional theory calculations (XAES-DFT). These parameters are related to cofactor assembly routes, metal site protonation, interactions between protein subunits, and initiation and gating of the ET.Our novel information from spectroscopy and theory should allow to assign prerequisites for the site-selective metal incorporation, compare structure-function relationships in RNR and oxidase enzymes, reveal the roles of cofactor species in the O2 activation and ET reactions, improve strategies for crystallography on the high-valent protein states, and construct detailed model structures for gaining deeper insight into the mechanisms of this fascinating manifold of dimetal cofactors.