Coupling CO2 Reduction and Acetyl-CoA Formation: The Role of a CO Capturing Tunnel in Enzymatic Catalysis.

The bifunctional CO-dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex couples the reduction of CO2 to the condensation of CO with a methyl moiety and CoA to acetyl-CoA. Catalysis occurs at two sites connected by a tunnel transporting the CO. In this study, we investigated how the bifunctional complex and its tunnel support catalysis using the CODH/ACS from Carboxydothermus hydrogenoformans as a model. Although CODH/ACS adapted to form a stable bifunctional complex with a secluded substrate tunnel, catalysis and CO transport is even more efficient when two monofunctional enzymes are coupled. Efficient CO channeling appears to be ensured by hydrophobic binding sites for CO, which act in a bucket-brigade fashion rather than as a simple tube. Tunnel remodeling showed that opening the tunnel increased activity but impaired directed transport of CO. Constricting the tunnel impaired activity and CO transport, suggesting that the tunnel evolved to sequester CO rather than to maximize turnover.

Stepwise O2 -Induced Rearrangement and Disassembly of the [NiFe4 (OH)(µ3 -S)4 ] Active Site Cluster of CO Dehydrogenase.

Ni,Fe-containing carbon monoxide dehydrogenases (CODHs) catalyze the reversible reduction of carbon dioxide to carbon monoxide. CODHs are found in anaerobic microorganisms and can rapidly lose their activity when exposed to air. What causes the loss of activity is unclear. In this study, we analyzed the time-dependent structural changes induced by the presence of air on the metal centers of CODH-II. We show that inactivation is a multistep process. In a reversible step, the open coordination site on the Ni ion is blocked by a Ni,Fe-bridging µ-sulfido or chlorido ligand. Blocking this open coordination site with a cyanide ligand stabilizes the cluster against O2 -induced decomposition, indicating that O2 attacks at the Ni ion. In the subsequent irreversible phase, nickel is lost, the Fe ions rearrange and the sulfido ligands disappear. Our data are consistent with a reversible reductive reactivation mechanism to protect CODHs from transient over-oxidation.

Structural basis for coupled ATP-driven electron transfer in the double-cubane cluster protein.

Electron transfers coupled to the hydrolysis of ATP allow various metalloenzymes to catalyze reductions at very negative reduction potentials. The double-cubane cluster protein (DCCP) catalyzes the reduction of small molecules, such as acetylene and hydrazine, with electrons provided by its cognate ATP-hydrolyzing reductase (DCCP-R). How ATP-driven electron transfer occurs is not known. To resolve the structural basis for ATP-driven electron transfer, we solved the structures of the DCCP:DCCP-R complex in three different states. The structures show that the DCCP-R homodimer is covalently bridged by a [4Fe4S] cluster that is aligned with the twofold axis of the DCCP homodimer, positioning the [4Fe4S] cluster to enable electron transfer to both double-cubane clusters in the DCCP dimer. DCCP and DCCP-R form stable complexes independent of oxidation state or nucleotides present, and electron transfer requires the hydrolysis of ATP. Electron transfer appears to be additionally driven by modulating the angle between the helices binding the [4Fe4S] cluster. We observed hydrogen bond networks running from the ATP binding site via the [4Fe4S] cluster in DCCP-R to the double-cubane cluster in DCCP, allowing the propagation of conformational changes. Remarkable similarities between the DCCP:DCCP-R complex and the nonhomologous nitrogenases suggest a convergent evolution of catalytic strategies to achieve ATP-driven electron transfers between iron-sulfur clusters.

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(H2 O)]-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.

Determining the oxidation state of elements by X-ray crystallography.

Protein-mediated redox reactions play a critical role in many biological processes and often occur at centres that contain metal ions as cofactors. In order to understand the exact mechanisms behind these reactions it is important to not only characterize the three-dimensional structures of these proteins and their cofactors, but also to identify the oxidation states of the cofactors involved and to correlate this knowledge with structural information. The only suitable approach for this based on crystallographic measurements is spatially resolved anomalous dispersion (SpReAD) refinement, a method that has been used previously to determine the redox states of metals in iron-sulfur cluster-containing proteins. In this article, the feasibility of this approach for small, non-iron-sulfur redox centres is demonstrated by employing SpReAD analysis to characterize Sulfolobus tokodaii sulerythrin, a ruberythrin-like protein that contains a binuclear metal centre. Differences in oxidation states between the individual iron ions of the binuclear metal centre are revealed in sulerythrin crystals treated with H2O2. Furthermore, data collection at high X-ray doses leads to photoreduction of this metal centre, showing that careful control of the total absorbed dose is a prerequisite for successfully determining the oxidation state through SpReAD analysis.

Bimetallic Mn, Fe, Co, and Ni Sites in a Four-Helix Bundle Protein: Metal Binding, Structure, and Peroxide Activation.

Bimetallic active sites in enzymes catalyze small-molecule conversions that are among the top 10 challenges in chemistry. As different metal cofactors are typically incorporated in varying protein scaffolds, it is demanding to disentangle the individual contributions of the metal and the protein matrix to the activity. Here, we compared the structure, properties, and hydrogen peroxide reactivity of four homobimetallic cofactors (Mn(II)2, Fe(II)2, Co(II)2, Ni(II)2) that were reconstituted into a four-helix bundle protein. Reconstituted proteins were studied in solution and in crystals. All metals bind with high affinity and yield similar cofactor structures. Cofactor variants react with H2O2 but differ in their turnover rates, accumulated oxidation states, and trapped peroxide-bound intermediates. Varying the metal composition thus creates opportunities to tune the reactivity of the bimetallic cofactor and to study and functionalize reactive species.

Nickel, Iron, Sulfur Sites.

Enzymes relying on the interplay of nickel, iron, and sulfur in their active sites are used by prokaryotes to catalyze reactions driving the global carbon and hydrogen cycles. The three enzymes, [NiFe] hydrogenases, Ni,Fe-containing carbon monoxide dehydrogenases and acetyl-CoA synthases share an ancient origin possibly derived from abiotic processes. Although their active sites have different compositions and assemble Ni, Fe, and S in different ways and for different purposes, they share a central role of Ni in substrate binding and activation, with sulfur linking the Ni ion to one or more Fe ions, which, although indispensable for function, supports the catalytic process in less understood ways. The review gives a short overview on the properties of the three individual enzymes highlighting their parallels and differences.

The two CO-dehydrogenases of Thermococcus sp. AM4.

Ni-containing CO-dehydrogenases (CODHs) allow some microorganisms to couple ATP synthesis to CO oxidation, or to use either CO or CO2 as a source of carbon. The recent detailed characterizations of some of them have evidenced a great diversity in terms of catalytic properties and resistance to O2. In an effort to increase the number of available CODHs, we have heterologously produced in Desulfovibrio fructosovorans, purified and characterized the two CooS-type CODHs (CooS1 and CooS2) from the hyperthermophilic archaeon Thermococcus sp. AM4 (Tc). We have also crystallized CooS2, which is coupled in vivo to a hydrogenase. CooS1 and CooS2 are homodimers, and harbour five metalloclusters: two [Ni4Fe-4S] C clusters, two [4Fe-4S] B clusters and one interfacial [4Fe-4S] D cluster. We show that both are dependent on a maturase, CooC1 or CooC2, which is interchangeable. The homologous protein CooC3 does not allow Ni insertion in either CooS. The two CODHs from Tc have similar properties: they can both oxidize and produce CO. The Michaelis constants (Km) are in the microM range for CO and in the mM range (CODH 1) or above (CODH 2) for CO2. Product inhibition is observed only for CO2 reduction, consistent with CO2 binding being much weaker than CO binding. The two enzymes are rather O2 sensitive (similarly to CODH II from Carboxydothermus hydrogenoformans), and react more slowly with O2 than any other CODH for which these data are available.

Double-Cubane [8Fe9S] Clusters: A Novel Nitrogenase-Related Cofactor in Biology.

Three different types of electron-transferring metallo-ATPases are able to couple ATP hydrolysis to the reduction of low-potential metal sites, thereby energizing an electron. Besides the Fe-protein known from nitrogenase and homologous enzymes, two other kinds of ATPase with different scaffolds and cofactors are used to achieve a unidirectional, energetic, uphill electron transfer to either reduce inactive Co-corrinoid-containing proteins (RACE-type activators) or a second iron-sulfur cluster-containing enzyme of a unique radical enzymes family (archerases). We have found a new cofactor in the latter enzyme family, that is, a double-cubane cluster with two [4Fe4S] subclusters bridged by a sulfido ligand. An enzyme containing this cofactor catalyzes the ATP-dependent reduction of small molecules, including acetylene. Thus, enzymes containing the double-cubane cofactor are analogous in function and share some structural features with nitrogenases.

X-ray Crystallography and Vibrational Spectroscopy Reveal the Key Determinants of Biocatalytic Dihydrogen Cycling by [NiFe] Hydrogenases.

[NiFe] hydrogenases are complex model enzymes for the reversible cleavage of dihydrogen (H2 ). However, structural determinants of efficient H2 binding to their [NiFe] active site are not properly understood. Here, we present crystallographic and vibrational-spectroscopic insights into the unexplored structure of the H2 -binding [NiFe] intermediate. Using an F420 -reducing [NiFe]-hydrogenase from Methanosarcina barkeri as a model enzyme, we show that the protein backbone provides a strained chelating scaffold that tunes the [NiFe] active site for efficient H2 binding and conversion. The protein matrix also directs H2 diffusion to the [NiFe] site via two gas channels and allows the distribution of electrons between functional protomers through a subunit-bridging FeS cluster. Our findings emphasize the relevance of an atypical Ni coordination, thereby providing a blueprint for the design of bio-inspired H2 -conversion catalysts.

Carbon Monoxide Dehydrogenases.

Carbon monoxide dehydrogenases (CODHs) catalyze the reversible oxidation of CO with water to CO2, two electrons, and two protons. Two classes of CODHs exist, having evolved from different scaffolds featuring active sites built from different transition metals. The basic properties of both classes are described in this overview chapter.

X-Ray Crystallography of Carbon Monoxide Dehydrogenases.

Carbon monoxide dehydrogenases (CODHs) are central players in the biogeochemical carbon monoxide (CO) cycle and have been extensively studied from the ecological level to the structural/molecular level. Of the two types of CODHs, the oxygen-tolerant CODHs use a bimetallic [CuSMo(=O)OH] center connected to the protein via a pyranopterin cofactor, whereas the oxygen-sensitive CODHs contain a [NiFe4S4-OHx]-cluster. Despite the fact that we have a basic understanding of how both types of CODHs use distinct active sites to catalyze the oxidation of CO with water to CO2, two protons, and two electrons (a reversible reaction in the cases of the oxygen-sensitive CODHs), many questions remain unanswered, especially concerning the electronic structures of the intermediate states. Since these states will likely be only revealed by the interplay of experimental and theoretical methods, there is a need to obtain accurate descriptions of the active site architectures in various states and, consequently, a need to generate crystals with good diffraction quality and collect data at element-specific wavelengths in order to determine the identity of elements in the case of mixed states. This chapter provides a description of the general working protocols for the crystallization and structural analysis of Cu,Mo-CODH and Ni,Fe-CODH that facilitates the mechanistic investigations of these important metalloenzymes.

Function and crystal structure of the dimeric P-loop ATPase CFD1 coordinating an exposed [4Fe-4S] cluster for transfer to apoproteins.

Maturation of iron-sulfur (Fe-S) proteins in eukaryotes requires complex machineries in mitochondria and cytosol. Initially, Fe-S clusters are assembled on dedicated scaffold proteins and then are trafficked to target apoproteins. Within the cytosolic Fe-S protein assembly (CIA) machinery, the conserved P-loop nucleoside triphosphatase Nbp35 performs a scaffold function. In yeast, Nbp35 cooperates with the related Cfd1, which is evolutionary less conserved and is absent in plants. Here, we investigated the potential scaffold function of human CFD1 (NUBP2) in CFD1-depleted HeLa cells by measuring Fe-S enzyme activities or 55Fe incorporation into Fe-S target proteins. We show that CFD1, in complex with NBP35 (NUBP1), performs a crucial role in the maturation of all tested cytosolic and nuclear Fe-S proteins, including essential ones involved in protein translation and DNA maintenance. CFD1 also matures iron regulatory protein 1 and thus is critical for cellular iron homeostasis. To better understand the scaffold function of CFD1-NBP35, we resolved the crystal structure of Chaetomium thermophilum holo-Cfd1 (ctCfd1) at 2.6-Å resolution as a model Cfd1 protein. Importantly, two ctCfd1 monomers coordinate a bridging [4Fe-4S] cluster via two conserved cysteine residues. The surface-exposed topology of the cluster is ideally suited for both de novo assembly and facile transfer to Fe-S apoproteins mediated by other CIA factors. ctCfd1 specifically interacted with ATP, which presumably associates with a pocket near the Cfd1 dimer interface formed by the conserved Walker motif. In contrast, ctNbp35 preferentially bound GTP, implying differential regulation of the two fungal scaffold components during Fe-S cluster assembly and/or release.

ATP-dependent substrate reduction at an [Fe8S9] double-cubane cluster.

Chemically demanding reductive conversions in biology, such as the reduction of dinitrogen to ammonia or the Birch-type reduction of aromatic compounds, depend on Fe/S-cluster-containing ATPases. These reductions are typically catalyzed by two-component systems, in which an Fe/S-cluster-containing ATPase energizes an electron to reduce a metal site on the acceptor protein that drives the reductive reaction. Here, we show a two-component system featuring a double-cubane [Fe8S9]-cluster [{Fe4S4(SCys)3}2(µ2-S)]. The double-cubane-cluster-containing enzyme is capable of reducing small molecules, such as acetylene (C2H2), azide (N3-), and hydrazine (N2H4). We thus present a class of metalloenzymes akin in fold, metal clusters, and reactivity to nitrogenases.

CODH-IV: A High-Efficiency CO-Scavenging CO Dehydrogenase with Resistance to O2.

CO dehydrogenases (CODHs) catalyse the reversible conversion between CO and CO2 . Genomic analysis indicated that the metabolic functions of CODHs vary. The genome of Carboxydothermus hydrogenoformans encodes five CODHs (CODH-I-V), of which CODH-IV is found in a gene cluster near a peroxide-reducing enzyme. Our kinetic and crystallographic experiments reveal that CODH-IV differs from other CODHs in several characteristic properties: it has a very high affinity for CO, oxidizes CO at diffusion-limited rate over a wide range of temperatures, and is more tolerant to oxygen than CODH-II. Thus, our observations support the idea that CODH-IV is a CO scavenger in defence against oxidative stress and highlight that CODHs are more diverse in terms of reactivity than expected.

Protein Dynamics in the Reductive Activation of a B12-Containing Enzyme.

B12-dependent proteins are involved in methyl transfer reactions ranging from the biosynthesis of methionine in humans to the formation of acetyl-CoA in anaerobic bacteria. During their catalytic cycle, they undergo large conformational changes to interact with various proteins. Recently, the crystal structure of the B12-containing corrinoid iron-sulfur protein (CoFeSP) in complex with its reductive activator (RACo) was determined, providing a first glimpse of how energy is transduced in the ATP-dependent reductive activation of corrinoid-containing methyltransferases. The thermodynamically uphill electron transfer from RACo to CoFeSP is accompanied by large movements of the cofactor-binding domains of CoFeSP. To refine the structure-based mechanism, we analyzed the conformational change of the B12-binding domain of CoFeSP by pulsed electron-electron double resonance and Förster resonance energy transfer spectroscopy. We show that the site-specific labels on the flexible B12-binding domain and the small subunit of CoFeSP move within 11 Å in the RACo:CoFeSP complex, consistent with the recent crystal structures. By analyzing the transient kinetics of formation and dissociation of the RACo:CoFeSP complex, we determined values of 0.75 µM-1 s-1 and 0.33 s-1 for rate constants kon and koff, respectively. Our results indicate that the large movement observed in crystals also occurs in solution and that neither the formation of the protein encounter complex nor the large movement of the B12-binding domain is rate-limiting for the ATP-dependent reductive activation of CoFeSP by RACo.

Carbon Monoxide Dehydrogenase Reduces Cyanate to Cyanide.

The biocatalytic function of carbon monoxide dehydrogenase (CODH) has a high environmental relevance owing to its ability to reduce CO2 . Despite numerous studies on CODH over the past decades, its catalytic mechanism is not yet fully understood. In the present combined spectroscopic and theoretical study, we report first evidences for a cyanate (NCO- ) to cyanide (CN- ) reduction at the C-cluster. The adduct remains bound to the catalytic center to form the so-called CN- -inhibited state. Notably, this conversion does not occur in crystals of the Carboxydothermus hydrogenoformans CODH enzyme (CODHIICh ), as indicated by the lack of the corresponding CN- stretching mode. The transformation of NCO- , which also acts as an inhibitor of the two-electron-reduced Cred2 state of CODH, could thus mimic CO2 turnover and open new perspectives for elucidation of the detailed catalytic mechanism of CODH.

Switchable Redox Chemistry of the Hexameric Tyrosine-Coordinated Heme Protein.

Hexameric tyrosine-coordinated heme protein HTHP from Silicibacter pomeroyi has been shown to exhibit peroxidase- and catalase-like activity. In the present study, detailed spectroscopic and electrochemical investigations were performed to analyze the redox properties and active site structure of HTHP. Potentiometric titration of HTHP in solution revealed a single redox transition at -0.54 V (vs Ag/AgCl), indicating six structurally identical tyrosine coordinates hemes. Cyclic voltammetry (CV) of immobilized HTHP afforded a distinctly more positive redox potential (-0.17 V) but failed to detect a transition at -0.54 V. Conversely, surface enhanced RR (SERR) spectroscopy provided evidence for both high- and low-potential transitions and for a partial loss of heme in the reduced state. The high-potential CV-active redox transition is attributed to the hemes of the barrel-shaped HTHP in a wheel-like orientation on the surface. Supported by coarse-grained simulations and SERR spectroscopy, the majority of HTHP is concluded to adopt a reverse-disc orientation, accounting for the low-potential transition. In view of the striking similarity of HTHP to the heme carriers HasA or HmbR regarding redox potential, Fe-Tyr ligation, and heme release, we propose heme transport as an alternative or additional function.

Ligand binding at the A-cluster in full-length or truncated acetyl-CoA synthase studied by X-ray absorption spectroscopy.

Bacteria integrate CO2 reduction and acetyl coenzyme-A (CoA) synthesis in the Wood-Ljungdal pathway. The acetyl-CoA synthase (ACS) active site is a [4Fe4S]-[NiNi] complex (A-cluster). The dinickel site structure (with proximal, p, and distal, d, ions) was studied by X-ray absorption spectroscopy in ACS variants comprising all three protein domains or only the C-terminal domain with the A-cluster. Both variants showed two square-planar Ni(II) sites and an OH- bound at Ni(II)p in oxidized enzyme and a H2O at Ni(I)p in reduced enzyme; a Ni(I)p-CO species was induced by CO incubation and a Ni(II)-CH3- species with an additional water ligand by a methyl group donor. These findings render a direct effect of the N-terminal and middle domains on the A-cluster structure unlikely.

AcsF Catalyzes the ATP-dependent Insertion of Nickel into the Ni,Ni-[4Fe4S] Cluster of Acetyl-CoA Synthase.

Acetyl-CoA synthase (ACS) catalyzes the reversible condensation of CO, CoA, and a methyl-cation to form acetyl-CoA at a unique Ni,Ni-[4Fe4S] cluster (the A-cluster). However, it was unknown which proteins support the assembly of the A-cluster. We analyzed the product of a gene from the cluster containing the ACS gene, cooC2 from Carboxydothermus hydrogenoformans, named AcsFCh, and showed that it acts as a maturation factor of ACS. AcsFCh and inactive ACS form a stable 2:1 complex that binds two nickel ions with higher affinity than the individual components. The nickel-bound ACS-AcsFCh complex remains inactive until MgATP is added, thereby converting inactive to active ACS. AcsFCh is a MinD-type ATPase and belongs to the CooC protein family, which can be divided into homologous subgroups. We propose that proteins of one subgroup are responsible for assembling the Ni,Ni-[4Fe4S] cluster of ACS, whereas proteins of a second subgroup mature the [Ni4Fe4S] cluster of carbon monoxide dehydrogenases.