The Ni-Fe-S C-cluster of carbon monoxide dehydrogenases (CODH), which catalyzes the reversible oxidation of CO to CO2, can be stabilized in four redox states: Cox, Cred1, Cint, and Cred2. The best-supported mechanism of catalysis involves a one-electron reductive activation of Cox to Cred1 and a catalytic cycle in which Cred1 binds and oxidizes CO, forming Cred2 and releasing CO2. Recently reported experiments appear to have disqualified this mechanism, as activation was concluded to require reduction to a C-cluster state more reduced than Cred1. The results presented in this dissertation suggest that the activation potential was milder than that required to reduce these clusters. The results support a mechanism in which Cred1 is the form of the cluster that reacts with CO. The structure of the active-site C-cluster in CO dehydrogenase from Carboxydothermus hydrogenoformans (CODHCh) includes a ??2-sulfide ion bridged to the Ni and unique Fe, while the same cluster in enzymes from Rhodospirillum rubrum (CODHRr) and Moorella thermoacetica (CODHMt) lack this ion. This difference was investigated by exploring effects of sulfide on activity and spectral properties. Sulfide partially inhibited CO oxidation activities of CODHRr and CODHMt. Adding sulfide to CODHMt in the Cred1 state caused the gav = 1.82 Electron Paramagnetic Resonance spectroscopy (EPR) signal to decline and new features to appear. Sulfide did not affect the gav = 1.86 signal from the Cred2 state. A model was developed in which sulfide binds reversibly to Cred1, inhibiting catalysis. The results also suggest that the substrate hydroxyl group bridges the Ni and unique Fe. A cysteine residue recently found to form a persulfide bond with the C-cluster was characterized. The Ser mutant of the persulfide-forming Cys316 was inactive and displayed no C-cluster EPR signals. Electronic absorption and metal analysis suggest that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or stability of the C-cluster, and/or for eliciting the redox chemistry of the C-cluster required for catalytic activity.

The Ni-Fe-S C-cluster of carbon monoxide dehydrogenases (CODH), which catalyzes the reversible oxidation of CO to CO2, can be stabilized in four redox states: Cox, Cred1, Cint, and Cred2. The best-supported mechanism of catalysis involves a one-electron reductive activation of Cox to Cred1 and a catalytic cycle in which Cred1 binds and oxidizes CO, forming Cred2 and releasing CO2. Recently reported experiments appear to have disqualified this mechanism, as activation was concluded to require reduction to a C-cluster state more reduced than Cred1. The results presented in this dissertation suggest that the activation potential was milder than that required to reduce these clusters. The results support a mechanism in which Cred1 is the form of the cluster that reacts with CO. The structure of the active-site C-cluster in CO dehydrogenase from Carboxydothermus hydrogenoformans (CODHCh) includes a ??2-sulfide ion bridged to the Ni and unique Fe, while the same cluster in enzymes from Rhodospirillum rubrum (CODHRr) and Moorella thermoacetica (CODHMt) lack this ion. This difference was investigated by exploring effects of sulfide on activity and spectral properties. Sulfide partially inhibited CO oxidation activities of CODHRr and CODHMt. Adding sulfide to CODHMt in the Cred1 state caused the gav = 1.82 Electron Paramagnetic Resonance spectroscopy (EPR) signal to decline and new features to appear. Sulfide did not affect the gav = 1.86 signal from the Cred2 state. A model was developed in which sulfide binds reversibly to Cred1, inhibiting catalysis. The results also suggest that the substrate hydroxyl group bridges the Ni and unique Fe. A cysteine residue recently found to form a persulfide bond with the C-cluster was characterized. The Ser mutant of the persulfide-forming Cys316 was inactive and displayed no C-cluster EPR signals. Electronic absorption and metal analysis suggest that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or stability of the C-cluster, and/or for eliciting the redox chemistry of the C-cluster required for catalytic activity.

This thesis describes the synthesis, characterisation and application of artificial metalloenzymes as catalysts. The focus was on two mutants of SCP-2L (SCP-2L A100C and SCP-2L V83C) both of which possess a hydrophobic tunnel in which apolar substrates can accumulate. The crystal structure of SCP-2L A100C was determined and discussed with a special emphasis on its hydrophobic tunnel. The SCP-2L mutants were covalently modified at their unique cysteine with two different N-ligands (phenanthroline or dipicolylamine based) or three different phosphine ligands (all based on triphenylphosphine) in order to increase their binding capabilities towards metals. The metal binding capabilities of these artificial proteins towards different transition metals was determined. Phenanthroline modified SCP-2L was found to be a promising scaffold for Pd(II)-, Cu(II)-, Ni(II)- and Co(II)-enzymes while dipicolylamine-modified SCP-2L was found to be a promising scaffold for Pd(II)-enzymes. The rhodium binding capacity of two additional phosphine modified protein scaffolds was also investigated. Promising scaffolds for Rh(I)- and Ir(I)-enzymes were identified. Rh-enzymes of the phosphine modified proteins were tested in the aqueous-organic biphasic hydroformylation of linear long chain 1-alkenes and compared to the Rh/TPPTS reference system. Some Rh-enzymes were found to be several orders of magnitude more active than the model system while yielding comparable selectivities. The reason for this remarkable reactivity increase could not be fully elucidated but several potential modes of action could be excluded. Cu-, Co-, and Ni-enzymes of N-ligand modified SCP-2L A100C were tested in the asymmetric Diels-Alder reaction between cyclopentadiene and trans-azachalcone. A promising 29% ee for the exo-product was found for the phenanthroline modified protein in the presence of nickel. Further improvement of these catalyst systems by chemical means (e.g. optimisation of ligand structure) and bio-molecular tools (e.g. optimisation of protein environment) can lead to even more active and (enantio)selective catalysts in the future.