Mechanism of Ni,Fe-containing Carbon monoxide Dehydrogenases

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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.