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

铁-氧超家族的特征是一个典型的高双金属FeFe辅因子，它的作用是激活O2-催化剂，涉及电子转移(ET)过程中的高价中间体。在所有生物体中对DNA合成至关重要的核糖核苷酸还原酶(RNRs)和对挑战转化反应很重要的配体结合氧化酶(RNRs)就是突出的例子。最近的研究结果表明，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，一个异双金属的MnFe物种形成了天然催化剂，而不是标准的FeFe辅因子，而且MnMn位点也是活跃的。此外，这些辅因子可以在标准RNR中存在和不存在众所周知的酪氨酸基的情况下发挥作用。这要求对这种类型的双金属辅因子的机理概念进行全面的重新评估。新的晶体结构代表着重大的进展，但只包含辅因子的低价状态。我们的目标是利用先进的X射线吸收和发射光谱技术结合密度泛函理论计算(XAES-DFT)来揭示Ct R2和Mt氧化酶蛋白中FeFe、MnFe和MnMn辅因子在O2激活的高价态(III2；III，IV；IV2)中的分子结构和电子构型。这些参数与辅因子组装途径、金属位置质子化、蛋白质亚基之间的相互作用以及ET的启动和门控有关。我们来自光谱和理论的新信息应该能够指定位置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，揭示辅因子物种在O2活化和ET反应中的作用，改进高价蛋白质状态的结晶学策略，并构建详细的模型结构，以更深入地了解这一迷人的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.