Mechanism of Ni,Fe-containing Carbon monoxide Dehydrogenases

含Ni、Fe的一氧化碳脱氢酶的机理

• 批准号: 206243590
• 负责人: Professor Dr. Holger Dobbek
• 金额: --
• 依托单位: Arbeitsgruppe Strukturbiologie und Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/206243590?language=en
• 关键词: Mechanism; Ni; Fe; containing; Carbon

Ni,Fe-containing carbon monoxide dehydrogenases (CODHs) catalyze the reversible oxidation of CO with water to CO2, two protons and two electrons, e.g. allowing bacteria and archaea to convert CO2 to CO, which is condensed with a methyl-cation and CoA to acetyl-CoA or conversely use CO as a source of electrons. Because CODHs require no overpotential in reducing CO2, the active site Ni,Fe-cluster of CODHs serves as a model for the design of chemical catalysts.In the last funding period, we (I) determined atomic resolution structures (dmin ~ 1.0 Å) of a Ni,Fe-CODH in complex with CO2 and cyanate and determined that the latter is also slowly reduced by CODHs. We could show that both substrate molecules bind as bridging ligands between Ni and Fe, are already reduced by two electrons and are stabilized by strong π-backbonding interactions with the Ni2+-ion. We also investigated the influence of key amino acids in the vicinity of the active site cluster C and determined their contribution to the kinetics of CO oxidation.(II) Furthermore, we recombinantly produced a before unobserved CODH (CODH-IVCh of Carboxydothermus hydrogenoformans), which is encoded in an oxidative stress operon. Its properties, a substantially higher tolerance to O2 and a very high affinity for CO, make it ideal for the organisms to use CO as source of electrons to reduce reactive oxygen species.(III) As a third project, we produced of protein annotated as a CODH, named CooSV, which shares the basic architecture of CODHs, but neither oxidizes CO nor reduces CO2 and contains a open asymmetric Fe/S cluster without Ni.In the next funding period, we want to (I) resolve open mechanistic questions of Ni,Fe-CODHs, like the early steps of CO and CO2 binding for which we will initiate XFEL measurements. Furthermore, we want to correlate the electronic and spatial structure linking the rich spectroscopic data on CODHs to defined structures of cluster C by combining single crystal EPR spectroscopy with X-ray diffraction. We also want to (II) investigate the substrate spectrum of the new enzyme, CooS-V, gain insight into its potential physiological role by investigating the enzyme with potential substrates in-vitro, in-vivo and in-crystallo. By extending the range of investigated enzymes, we want to gain insights not only on the structure and mechanism, but also the diversity and evolution of CODHs.

含 Ni、Fe 的一氧化碳脱氢酶 (CODH) 催化 CO 与水可逆氧化为 CO2、两个质子和两个电子，例如允许细菌和古细菌将 CO2 转化为 CO，然后与甲基阳离子和 CoA 缩合为乙酰 CoA，或者相反地使用 CO 作为电子源。由于 CODH 不需要还原 CO2 的过电势，因此 CODH 的活性位点 Ni,Fe-簇可以作为化学催化剂设计的模型。在上一个资助期间，我们 (I) 确定了 Ni,Fe-CODH 与 CO2 和氰酸盐复合物的原子分辨率结构 (dmin ~ 1.0 Å)，并确定后者也能被 CODH 缓慢还原。我们可以证明，两种底物分子都作为 Ni 和 Fe 之间的桥配体结合，已经被两个电子还原，并通过与 Ni2+ 离子的强 π 背键相互作用而稳定。我们还研究了活性位点簇C附近的关键氨基酸的影响，并确定了它们对CO氧化动力学的贡献。 (II)此外，我们重组产生了以前未观察到的CODH（Carboxydothermus Hydronoformans的CODH-IVCh），它由氧化应激操纵子编码。它对 O2 具有更高的耐受性，对 CO 具有极高的亲和力，使其非常适合生物体使用 CO 作为电子源来减少活性氧。(III) 作为第三个项目，我们生产了注释为 CODH 的蛋白质，命名为 CooSV，它具有 CODH 的基本结构，但既不氧化 CO 也不还原 CO2，并且包含开放的不对称 Fe/S 簇，不 Ni。在下一个资助期间，我们希望 (I) 解决 Ni,Fe-CODH 的开放机制问题，例如 CO 和 CO2 结合的早期步骤，我们将为此启动 XFEL 测量。此外，我们希望通过将单晶 EPR 光谱与 X 射线衍射相结合，将 CODH 上丰富的光谱数据与簇 C 的定义结构联系起来的电子和空间结构相关联。我们还想 (II) 研究新酶 CooS-V 的底物谱，通过在体外、体内和晶体内研究具有潜在底物的酶，深入了解其潜在的生理作用。通过扩大研究酶的范围，我们不仅希望深入了解 CODH 的结构和机制，还希望了解 CODH 的多样性和进化。