- Carbon monoxide dehydrogenase (1) (remove)
- Mechanism of carbon monoxide oxidation at the active site [Ni-4Fe-5S] cluster of carbon monoxide dehydrogenase from Carboxydothermus hydrogenoformans (2009)
- The carbon monoxide (CO) metabolism relies on CO-dehydrogenase (CODH) that oxidizes CO with H2O to CO2. The crystal structures of the native Ni-Fe CODHIICh from the CO-grown thermophilic hydrogenogenic anaerobic bacterium Carboxydothermus hydrogenoformans reveal the active site as a [Ni-4Fe-5S] cluster C, carrying a bimetallic Ni-(mu2S)-Fe1 subsite with a bridging mu2S. This is the assumed site, where the oxidation of CO occurs. However, cluster C in other catalytically active Ni-Fe CODHs, from Rhodospirillum rubrum, Moorella thermoacetica, and recombinant CODHIICh expressed in Escherichia coli, for example, lack the ¥ì2S bridging Ni and Fe and contain a [Ni-4Fe-4S] form of a cluster C. The CO oxidation mechanism proposed based on crystallographical and biochemical studies involved the apical binding of CO at the nickel ion and the activation of water at the Fe1 ion of the cluster. In order to understand how CO interacts with the active site of native CODHIICh and what function does the bridging mu2S ligand fulfill in the enzyme, this research work focuses (i) on the interaction of cluster C with CO analogue potassium cyanide and analysis of the resulting type of nickel coordination and (ii) on the effect of sodium sulfide on the enzymatic activities of the native CODHIICh. Under catalytic conditions, cyanide acts as a competitive inhibitor of CODHIICh with respect to CO. Under N2 gas phase without electron acceptor (non-catalytic conditions), cyanide inhibits CODHIICh at highly reduced state (low redox-potentials - 500 mV), not at oxidized and slightly reduced (- 320 mV) states. Cyanide is not able to inhibit CODHIICh at reduced conditions (- 500 mV) when CO is present in the atmosphere. Therefore, the interaction of cyanide with reduced active site is expected to mimic the substrate. The binding of cyanide to the nickel ion has been discovered by x-ray absorption spectroscopy and confirmed by x-ray crystallography at the atomic resolution. This structure comprises an intermediate state of cluster C with CO in NiFe-CODHIICh. In this reaction, cyanide displaces the mu2S ligand giving rise to square-planar Ni with three S and one CN ligands. Cluster C and its protein environment undergo significant conformational changes induced by the binding of cyanide. Remarkably, Fe1 is displaced by 1,1 A, which reduces the Fe1 to Ni distance by 0,1 A. Electron densities of the CN ligand estimate an occupancy of 80 % and N atom of cyanide is in hydrogen bonding distance to His93 and Lys563, which are involved in proton transfer network. The binding of cyanide eliminates the bridging ¥ì2S from the cluster C yielding H2S, whereas the Ni-(mu2S)-Fe1 bridge is reformed after the catalytic cycle. It is likely that the high rate of CO oxidation (Kcat/Km of 1.7 x 109 M-1 s-1 at 70 ¡ÆC) and subsequent rebinding of mu2S would prevent the release of this ion from the protein by diffusion-controlled process (108 to 109 M-1 s-1). Cyanide-inhibited reduced CODHIICh is fully reactivated after the release of cyanide upon incubation at 70 ¡ÆC in the presence of low-potential reductants. The square-planar NiS4-coordinated cluster C is recovered by reactivation with sulfide, resulting in fully active enzyme, which includes the reformation of the Ni-(mu2S)-Fe1 bridge. Reactivation in the absence of sulfide generates the NiS3-coordination, lacking the mu2S ligand, and results in partially active enzyme. NiS3-coordinated cluster C is readily converted to NiS4-conformation by incorporation of sulfide. This conversion results in an increase of CO oxidation activity and a stabilization of cluster C at growth temperatures of the bacterium. In addition, the NiS4-conformation displays better catalytic efficiency (Kcat/Km) than NiS3 under low concentrations of CO. Inhibition of CO2 reduction activity by sulfide and higher CO2 reduction activity of NiS3-conformation suggest that the mu2S ligand retards the binding of CO2 to Ni. The crystal structure of CO2 loaded NiS3-cluster further convinces this assumption. The mu2S ligand accelerates the physiologically relevant CO oxidation, prevents inhibition by the product CO2, and inhibits a non-physiological CO2 reduction. These functions of the bridging mu2S are of physiologically importance for the metabolism of C. hydrogenoformans in highly reducing, CO-limited, and CO2-rich volcanic environments. Thus, it is concluded that the [Ni-4Fe-5S]-form of cluster C is the biologically relevant species in CODHIICh. A CO oxidation mechanism developed in this thesis is based on the structures of cyanide-bound intermediated state and CO2-bound state, as well as on the kinetic data on the reactivity of the enzyme with CO, potassium cyanide and sodium sulfide.