Mechanism of Ni,Fe-containing Carbon monoxide Dehydrogenases

含Ni、Fe的一氧化碳脱氢酶（CODH）催化CO与水可逆氧化成CO2、两个质子和两个电子，例如允许细菌和古细菌将CO2转化成CO，其与甲基阳离子和CoA缩合成乙酰CoA，或者相反地使用CO作为电子源。由于CODHs在还原CO2时不需要过电位，因此CODHs的活性中心Ni，Fe-簇可作为化学催化剂设计的模型。在上一个资助期内，我们（I）测定了Ni，Fe-CODHs与CO2和氰酸盐络合物的原子分辨率结构（dmin ~ 1.0），并确定后者也被CODHs缓慢还原。我们可以证明，两种底物分子作为Ni和Fe之间的桥接配体结合，已经被两个电子还原，并且通过与Ni 2+离子的强π-背键相互作用而稳定。我们还研究了活性中心簇C附近的关键氨基酸的影响，并确定它们对CO氧化动力学的贡献。(II)此外，我们重组产生了之前未观察到的CODH（CODH-IVCh的Carboxydothermus hydrogenoformans），这是编码的氧化应激操纵子。它的特性，对O2的耐受性明显更高，对CO的亲和力非常高，使其成为生物体使用CO作为电子源以减少活性氧的理想选择。(III)作为第三个项目，我们生产的蛋白质注释为CODH，命名为CooSV，它共享CODH的基本结构，但既不氧化CO也不还原CO2，并包含一个开放的不对称Fe/S簇，没有Ni。在下一个资助期内，我们希望（I）解决Ni，Fe-CODH的开放机制问题，如CO和CO2结合的早期步骤，我们将启动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的定义结构相关联。我们还想（II）研究新酶CooS-V的底物谱，通过研究酶与体外、体内和晶体内潜在底物的关系，深入了解其潜在的生理作用。通过扩大研究酶的范围，我们不仅希望了解CODH的结构和机制，还希望了解CODH的多样性和进化。

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.