Coupling CO 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 CO 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.

Stepwise O -Induced Rearrangement and Disassembly of the [NiFe (OH)(μ -S) ] 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.

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.

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

Ni,Fe-containing carbon monoxide dehydrogenases (CODHs) catalyze the reversible reduction of CO 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-V .

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.

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), Fe(II), Co(II), Ni(II)) that were reconstituted into a four-helix bundle protein.

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 CO 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 O. 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.

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.

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 (H ). However, structural determinants of efficient H binding to their [NiFe] active site are not properly understood. Here, we present crystallographic and vibrational-spectroscopic insights into the unexplored structure of the H -binding [NiFe] intermediate.

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 [NiFeS-OH]-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 CO, 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.

Carbon Monoxide Dehydrogenases.

Carbon monoxide dehydrogenases (CODHs) catalyze the reversible oxidation of CO with water to CO, 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.

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.

ATP-dependent substrate reduction at an [FeS] 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 [FeS]-cluster [{FeS(SCys)}(-S)].

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

CO dehydrogenases (CODHs) catalyse the reversible conversion between CO and CO . 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.

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.

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 CO . 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.

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.

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.

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.

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.

Molecularly Imprinted Electropolymer for a Hexameric Heme Protein with Direct Electron Transfer and Peroxide Electrocatalysis.

For the first time a molecularly imprinted polymer (MIP) with direct electron transfer (DET) and bioelectrocatalytic activity of the target protein is presented. Thin films of MIPs for the recognition of a hexameric tyrosine-coordinated heme protein (HTHP) have been prepared by electropolymerization of scopoletin after oriented assembly of HTHP on a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold electrodes. Cavities which should resemble the shape and size of HTHP were formed by template removal.

Quercetin 2,4-Dioxygenase Activates Dioxygen in a Side-On O2-Ni Complex.

Quercetin 2,4-dioxygenase (quercetinase) from Streptomyces uses nickel as the active-site cofactor to catalyze oxidative cleavage of the flavonol quercetin. How this unusual active-site metal supports catalysis and O2 activation is under debate. We present crystal structures of Ni-quercetinase in three different states, thus providing direct insight into how quercetin and O2 are activated at the Ni(2+) ion.

Structure of an Actinobacterial-Type [NiFe]-Hydrogenase Reveals Insight into O2-Tolerant H2 Oxidation.

A novel group of bacterial [NiFe]-hydrogenases is responsible for high-affinity H2 uptake from the troposphere, and is therefore thought to play an important role in the global H2 cycle. Here we present the first crystal structure at 2.85-Å resolution of such an actinobacterial-type hydrogenase (AH), which was isolated from the dihydrogen oxidizing bacterium, Ralstonia eutropha.

How the [NiFe4S4] Cluster of CO Dehydrogenase Activates CO2 and NCO(-).

Ni,Fe-containing CO dehydrogenases (CODHs) use a [NiFe4S4] cluster, termed cluster C, to reversibly reduce CO2 to CO with high turnover number. Binding to Ni and Fe activates CO2, but current crystal structures have insufficient resolution to analyze the geometry of bound CO2 and reveal the extent and nature of its activation. The crystal structures of CODH in complex with CO2 and the isoelectronic inhibitor NCO(-) are reported at true atomic resolution (dmin ≤1.