A Morphing [4Fe-3S-nO]-Cluster within a Carbon Monoxide Dehydrogenase Scaffold

Ni,Fe-containing carbon monoxide dehydrogenases (CODHs) catalyze the reversible reduction of CO2 to CO. Several anaerobic microorganisms encode multiple CODHs in their genome, of which some, despite being annotated as CODHs, lack a cysteine of the canonical binding motif for the active site Ni,Fe-cluster. Here, we report on the structure and reactivity of such a deviant enzyme, termed CooS-VCh. Its structure reveals the typical CODH scaffold, but contains an iron-sulfur-oxo hybrid-cluster. Although closely related to true CODHs, CooS-VCh catalyzes neither CO oxidation, nor CO2 reduction. The active site of CooS-VCh undergoes a redox-dependent restructuring between a reduced [4Fe-3S]-cluster and an oxidized [4Fe-2S-S*-2O-2(H2O)]-cluster. Hydroxylamine, a slow-turnover substrate of CooS-VCh, oxidizes the hybrid-cluster in two structurally distinct steps. Overall, minor changes in CODHs are sufficient to accommodate a Fe/S/O-cluster in place of the Ni,Fe-heterocubane-cluster of CODHs

Biochemische und strukturelle Untersuchungen der Kohlenmonoxid-Dehydrogenasen CODH-II und CODH-V aus Carboxydothermus hydrogenoformans

Eine Vielzahl strikt anaerober Organismen verwendet den reduktiven Acetyl-CoA-Weg zum autotrophen Wachstum mit Kohlenmonoxid als einziger Kohlenstoffquelle. Die Kohlenmonoxid-Dehydrogenase (CODH) ist das Schlüsselenzym dieses Stoffwechselweges und katalysiert die Oxidation von CO mit Raten von bis zu 31,000 s–1 und die Reduktion von CO2 mit bis zu 12 s–1 an einem [Ni4Fe4S-OHx]-Cluster (C-Cluster). Das Genom des thermophilen und hydrogenogenen Bakteriums Carboxydothermus hydrogenoformans enthält insgesamt fünf Gene, die für CODHs kodieren. Anhand der Genumgebung wurden dabei unterschiedliche Rollen für die einzelnen CODHs vorgeschlagen. Für ein besseres Verständnis der molekularen Prozesse in der Katalyse, wurden CODH-IICh und -VCh heterolog in Escherichia coli produziert und biochemisch und strukturell charakterisiert.A variety of strict anaerobic organisms employ the reductive acetyl-CoA path for autotrophic growth, using carbon monoxide as sole carbon source. Carbon monoxide dehydrogenase (CODH) is the key enzyme of the path and catalyzes CO oxidation with rates of 31,000 s–1 and CO2 reduction with rates of 12 s–1 at a [Ni4Fe4S-OHx] cluster (cluster C). The genome of the thermophilic and hydrogenogenic bacterium Carboxydothermus hydrogenoformans contains five copies of genes coding for the catalytic subunit of a CODH. According to the gene environment, different physiological roles for the individual CODHs were proposed. To compare their respective structure and catalytic function, CODH-IICh and -VCh were heterologously produced in Escherichia coli and biochemically and structurally investigated

A Morphing [4Fe-3S-nO]-Cluster within a Carbon Monoxide Dehydrogenase Scaffold

Ni,Fe-containing carbon monoxide dehydrogenases (CODHs) catalyze the reversible reduction of CO2 to CO. Several anaerobic microorganisms encode multiple CODHs in their genome, of which some, despite being annotated as CODHs, lack a cysteine of the canonical binding motif for the active site Ni,Fe-cluster. Here, we report on the structure and reactivity of such a deviant enzyme, termed CooS-VCh. Its structure reveals the typical CODH scaffold, but contains an iron-sulfur-oxo hybrid-cluster. Although closely related to true CODHs, CooS-VCh catalyzes neither CO oxidation, nor CO2 reduction. The active site of CooS-VCh undergoes a redox-dependent restructuring between a reduced [4Fe-3S]-cluster and an oxidized [4Fe-2S-S*-2O-2(H2O)]-cluster. Hydroxylamine, a slow-turnover substrate of CooS-VCh, oxidizes the hybrid-cluster in two structurally distinct steps. Overall, minor changes in CODHs are sufficient to accommodate a Fe/S/O-cluster in place of the Ni,Fe-heterocubane-cluster of CODHs.Peer Reviewe

When the inhibitor tells more than the substrate: the cyanide-bound state of a carbon monoxide dehydrogenase

Carbon monoxide dehydrogenase (CODH) is a key enzyme for reversible CO interconversion. To elucidate structural and mechanistic details of CO binding at the CODH active site (C-cluster), cyanide is frequently used as an iso-electronic substitute and inhibitor. However, previous studies revealed conflicting results on the structure of the cyanide-bound complex and the mechanism of cyanide-inhibition. To address this issue in this work, we have employed IR spectroscopy, crystallography, site directed mutagenesis, and theoretical methods to analyse the cyanide complex of the CODH from Carboxydothermus hydrogenoformans (CODHIICh). IR spectroscopy demonstrates that a single cyanide binds to the Ni ion. Whereas the inhibitor could be partially removed at elevated temperature, irreversible degradation of the C-cluster occurred in the presence of an excess of cyanide on the long-minute time scale, eventually leading to the formation of [Fe(CN)(6)](4-) and [Ni(CN)(4)](2-) complexes. Theoretical calculations based on a new high-resolution structure of the cyanide-bound CODHIICh indicated that cyanide binding to the Ni ion occurs upon dissociation of the hydroxyl ligand from the Fe-1 subsite of the C-cluster. The hydroxyl group is presumably protonated by Lys563 which, unlike to His93, does not form a hydrogen bond with the cyanide ligand. A stable deprotonated 3-amino group of Lys563 in the cyanide complex is consistent with the nearly unchanged C equivalent to N stretching in the Lys563Ala variant of CODHIICh. These findings support the view that the proton channel connecting the solution phase with the active site displays a strict directionality, controlled by the oxidation state of the C-cluster.DFG, EXC 314, Unifying Concepts in Catalysi