Science 30 November 2007:
Vol. 318. no. 5855, pp. 1461 - 1464
DOI: 10.1126/science.1148481

 Carbon Dioxide Activation at the Ni,Fe-Cluster of Anaerobic Carbon Monoxide Dehydrogenase

Anaerobic CO dehydrogenases catalyze the reversible oxidation of CO to CO₂ at a complex Ni-, Fe-, and S-containing metal center called cluster C. We report crystal structures of CO dehydrogenase II from Carboxydothermus hydrogenoformans in three different states. In a reduced state, exogenous CO₂ supplied in solution is bound and reductively activated by cluster C. In the intermediate structure, CO₂ acts as a bridging ligand between Ni and the asymmetrically coordinated Fe, where it completes the square-planar coordination of the Ni ion. It replaces a water/hydroxo ligand bound to the Fe ion in the other two states. The structures define the mechanism of CO oxidation and CO₂ reduction at the Ni-Fe site of cluster C.

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Laboratorium Proteinkristallographie and Forschungszentrum für Bio-Makromoleküle, Universität Bayreuth, D-95440 Bayreuth, Germany.

^* To whom correspondence should be addressed. E-mail: holger.dobbek@uni-bayreuth.de

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The biological redox transformations of CO₂, N₂, and H₂ are essential processes in global biogeochemical cycles and are catalyzed by enzymes containing complex metal clusters based on iron and sulfur whose detailed function is still poorly understood (1, 2). Carbon monoxide dehydrogenases (CODHases) are the biological catalysts for the reversible oxidation of CO to CO₂, with water as the source of oxygen: CO + H₂O CO₂ +2e^– +2H⁺ (Eq. 1). Two principal types of CODHases have been described that differ in their cofactor composition, structure, and stability in the presence of dioxygen: Anaerobic bacteria and archaea use oxygen-sensitive Ni- and Fe-containing CODHases, whereas aerobic, carboxydotrophic bacteria use a Cu-, Mo-, and Fe-containing flavoenzyme (3). The Ni,Fe-CODHases are monofunctional or bifunctional enzymes associated with Ni,Fe-containing acetyl–coenzyme A (CoA) synthases (ACS) (4, 5)[for review, see (3, 6)].

CO oxidation and CO₂ reduction at the active site, cluster C, of Ni,Fe-CODHases are proposed to require three different oxidation states differing by one electron (C_red1, C_int, and C_red2) (6). In this model, the C_red1 state of cluster C converts CO to CO₂ and is formed at redox potentials below –200 mV (7). At pH = 7.0, the midpoint potential for the conversion of C_red1 to C_red2 is –530 mV (8, 9), similar to the normal potential of the CO₂-CO couple of –558 mV (10). Consequently, C_red2 is generated by a two-electron reduction of the C_red1 state via C_int (11). The structure of cluster C was revealed by crystallographic analysis of CODHases isolated from Carboxydothermus hydrogenoformans (CODHII_Ch) and Rhodospirillum rubrum (CODH_Rr) at 1.6 and 2.8 Å resolution, respectively (12, 13). Cluster C in CODHII_Ch has been described as an asymmetric [NiFe₄S₅] center, which comprises an integral Ni ion coordinated by four sulfur ligands with square-planar geometry (12). An asymmetrically coordinated Fe ion (Fe1) is found close to the Ni ion. In CO-treated CODH_Rr, cluster C has a similar structure with a cubane [NiFe₃S₄] center linked to a mononuclear Fe site (13). Corresponding structures of cluster C were also identified in the crystal structures of bifunctional ACS/CODH isolated from Moorella thermoacetica (CODH_Mt) (4, 5). Mechanisms proposed for the reversible oxidation of CO to CO₂ posit the activation of H₂O and CO as well as the stabilization of a metal-bound COO(H) intermediate. However, the structure of these states remained elusive. We describe how H₂O and CO₂ are bound and activated and propose a structure-based model for CO oxidation and CO₂ reduction at the Ni-Fe1 site of cluster C.

An expression system for CODHII_Ch established in Escherichia coli enables a one-step purification of active enzyme. Crystals of recombinant CODHII_Ch diffract up to 1.40 Å resolution on a rotating anode x-ray generator (table S1). The overall structure of recombinant CODHII_Ch is identical to the structure of native CODHII_Ch (12). CODHII_Ch crystals were held at a defined redox potential of –600 mV for 3 hours with use of Ti(III) citrate. These crystals were either directly frozen in liquid nitrogen, generating the –600 mV state; oxidized via incubation with methylviologen (MV_ox) and dithiothreitol (DTT) and then incubated with DTT to give the –320 mV state; or incubated in the –600 mV solution with NaHCO₃ as the CO₂ source, generating the –600 mV+CO₂ state. The –600 mV state (equivalent in its redox potential to the C_red2 state) and the –320 mV state (equivalent to the C_red1 state) display practically identical structures for cluster C (Fig. 1, A and C). In both structures, the Ni ion is coordinated by three sulfur ligands with distorted T-shaped coordination geometry (Fig. 1, A and C, and table S3). Fe1 is coordinated by His²⁶¹ (H261), Cys²⁹⁵ (C295), a µ₃-sulfido ligand, and a monoatomic ligand (Fig. 1, A and C, and figs. S2 and S4). A weakly occupied alternative position for Fe1 (Fe1B) is observed in both states (Fig. 1 and table S2). The monoatomic ligand is a distance of 2.7 Å from the Ni ion and occupies the position that would complete the square-planar coordination of the Ni. The electron density of the ligand can be modeled as a light atom (C, N, or O) with occupancies of about 60 to 70% or a sulfur atom with 30% occupancy. The observed Fe-ligand bond lengths of 1.93 to 1.95 Å are atypical for Fe-S bonds, whereas a H₂O/OH^– ligand is consistent with the refined bond length, the relative occupancies of ligand and Fe1 (table S2), and spectroscopic investigations of the C_red1 state. The long distance between Ni and the ligand suggests a weak Ni-OH_x interaction. A H₂O/OH^– ligand has been detected bound to a high spin Fe²⁺ ion called ferrous component II (FCII) in the C_red1 state (14, 15), so the crystal structure is consistent with Fe1 in the –320 mV state being FCII.

In the presence of appropriate reducing agents, Ni,Fe-CODHases can catalyze the reduction of CO₂ (16). The structure of CODHII_Ch with CO₂ (–600 mV+CO₂ state) reveals a triatomic ligand bridging Ni and Fe1, which replaces the water/hydroxo ligand at Fe1 (Fig. 1B). Modeling the ligand as CO₂ fully satisfies the observed electron density maps, whereas modeling with one or two atoms does not. CO₂ bound to cluster C acts as a ¹ OCO ligand at Ni²⁺ with a Ni-C distance of 1.96 Å and completes the square-planar coordination geometry typically found for Ni²⁺ ions. CO₂ acts as a ¹ OCO ligand at Fe1 with an Fe1-O1 distance of 2.05 Å, resulting in a µ₂-² binding mode of CO₂ bridging the Ni-Fe1 site (Fig. 1B). Like the H₂O/OH^– ligand in the –600 mV and –320 mV states, O1 of CO₂ is in hydrogen-bonding distance to Lys⁵⁶³ (K563) (Fig. 1B). O2 is in hydrogen-bonding distance to His⁹³. CO₂ binding to cluster C causes only minor changes in the geometry of the cluster (Fig. 2). The change of the distorted T-shaped to the square-planar coordination at the Ni ion induces a small shift in the Ni position of about 0.2 Å and widens the Cys⁵²⁶S-Ni-S₃ angle (table S3).

The ability of CO₂ to coordinate transition metal complexes is well documented (17). Coordination of CO₂ at the carbon atom results in a net electron transfer from the metal into the antibonding lowest unoccupied molecular orbital of CO₂. This activation of CO₂ increases the negative partial charges at the oxygen atoms, which are stabilized by binding to electron-deficient centers like transition metals or by forming hydrogen bonds (17). In the cluster C-CO₂ complex, Ni acts as the Lewis base, and Fe1 is the Lewis acid that together with K563 stabilizes the negative partial charge on O1. The deviation from linearity along the O-C-O axis [O-C-O 133° (table S3)] is in agreement with the activation of CO₂ by binding to cluster C.

In previous structural characterizations of native preparations of CODHII_Ch, we identified a µ-sulfido ligand (S2) bridging Ni and Fe1 (12, 18). The enzyme used for crystallization as well as dissolved crystals had high specific activities of 14.000 units mg^–1, and on the basis of a positive correlation between the presence of the S2 ligand and enzyme activity it was postulated that S2 is essential for the catalytic CO oxidation (18, 19). However, the necessity of the S2 ligand for catalysis was debated when sulfide was shown to reversibly inactivate CODH_Rr and CODH_Mt, leading to short lag phases (20), and no S2 ligands were identified in the crystal structures of CODH_Rr and CODH_Mt (5, 13). Here, we describe the structures of [NiFe₄S₄(OH_x)/(CO₂)] clusters without S2 ligand in crystals with high specific CO oxidation activities (11.000 to 13.500 units mg^–1), showing that the presence of S2 is not necessary for catalysis. Furthermore, the S2 ligand occupies the binding site of two substrates of Ni,Fe-CODHases, water, and CO₂. The H₂O/OH^– ligand identified requires the same coordination site at Fe1 as the bridging S2 ligand (fig. S5), and CO₂ binds to the two open coordination sites of Ni and Fe1. Thus, we suggest that the S2 ligand between Ni and Fe1 is absent in catalytically competent enzyme species and can be reductively or chemically replaced, activating the enzyme.

The three presented structures offer direct insight into the reaction mechanism (Fig. 3). The [NiFe₄S₄OH_x] cluster determined in the –320 mV state is the functional state that activates CO and contains the H₂O/OH^– ligand. The Ni²⁺ ion is positioned at the end of the substrate channel, and its three sulfido ligands act as donors to the metal, enabling CO to bind to the Ni²⁺ ion (21). The Ni²⁺ ion has two open coordination sites, allowing either an apical binding of CO to form a distorted tetrahedral geometry or CO binding equatorially to complete the square-planar coordination geometry. Binding of CO in the apical position has been proposed for CO-treated crystals of CODH_Rr and CODH_Mt (5, 13). Modeling of CO in the apical position places the CO-carbon atom more than 3.5 Å apart from the H₂O/OH^– ligand and makes further rearrangements necessary for the reaction to proceed. In contrast, CO binding to complete a distorted square-planar coordination of the Ni²⁺ ion results in a OC-OH_x distance of less than 2 Å (Fig. 3, step II). The binding of CO to a weakly backdonating metal like Ni²⁺ results in an electrophilic carbon atom and facilitates its reaction with the Fe1-bound H₂O/OH^– ligand to a metal-carboxylate species as observed in the –600 mV+CO₂ state (Figs. 1B and 3, step III). Product release may be assisted by the reversible ligand exchange of CO₂ against H₂O at Fe1 and is accompanied by a two-electron reduction of cluster C, generating the C_red2 state. The H₂O/OH^– ligand can be replenished by a neighboring network of solvent molecules (fig. S6). A comparison of the [NiFe₄S₄OH_x]and [NiFe₄S₄(CO₂)] states reveals the positions of Ni and Fe1 to be largely unaffected by the presence or absence of the CO₂ ligand (Fig. 2). The [Fe₃S₄] site of cluster C provides a solid metal-sulfur frame in which Ni and Fe1 are held in place and serves as an electronic buffer to compensate for the electronic changes at Ni and Fe1 during the catalytic cycle. The small structural changes of cluster C agree well with the low reorganization energy expected for a reaction with turnover rates of 31,000 s^–1 and a ratio of k_cat (catalytic rate constant)/K_m (Michaelis constant) of 1.7 x 10⁹ M^–1 s^–1 at +70°C (22).

The structure-based mechanism outlined agrees in all central aspects with the bimetallic mechanism proposed on the basis of electron paramagnetic resonance (EPR), electron nuclear double-resonance (ENDOR), and Mössbauer spectroscopy (14, 15).

References and Notes

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Carbon Dioxide Activation at the Ni,Fe-Cluster of Anaerobic Carbon Monoxide Dehydrogenase

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