Jae-Hun Jeoung

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.

Structural basis of cyanide inhibition of Ni, Fe-containing carbon monoxide dehydrogenase.

Carbon monoxide dehydrogenases (CODHs) catalyze the reversible oxidation of carbon monoxide with water to carbon dioxide, two protons, and two electrons. The CODHs of anaerobic microorganisms harbor a complex Ni/Fe/S-containing metal center called a C-cluster in their active site, which activates the substrates water and carbon monoxide, stabilizes an intermediary metal-carboxylate, and transiently stores the two electrons generated in the reaction. Several small molecules have been reported to inhibit carbon monoxide oxidation by CODHs, among which the cyanide anion acts as a slow binding inhibitor. Cyanide is isoelectronic to the substrate carbon monoxide, and its binding to the C-cluster has been reported to involve nickel, nickel and iron, or only iron. We report the crystal structure of CODH-II from Carboxydothermus hydrogenoformans in complex with cyanide at 1.36 A resolution. The structure reveals that cyanide binds to the C-cluster at an open coordination site completing the square-planar coordination geometry of the nickel ion. While active CODH has a water/hydroxo-ligand bound to an iron ion near nickel, in the cyanide complex the water/hydroxo-ligand is lost and iron occupies a position more close to the nickel ion. Based on the structure, we suggest that the competitive inhibitory character of cyanide originates from it obstruction of carbon monoxide binding to the nickel ion while the slow binding inhibition is due to a conformational change of the protein during which the water/hydroxo-ligand bound to iron is lost.

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 ≤1.1 Å). Like CO2, NCO(-) is a μ2,η(2) ligand of the cluster and acts as a mechanism-based inhibitor. While bound CO2 has the geometry of a carboxylate group, NCO(-) is transformed into a carbamoyl group, thus indicating that both molecules undergo a formal two-electron reduction after binding and are stabilized by substantial π backbonding. The structures reveal the combination of stable μ2,η(2) coordination by Ni and Fe2 with reductive activation as the basis for both the turnover of CO2 and inhibition by NCO(-).

Visualizing the substrate-, superoxo-, alkylperoxo-, and product-bound states at the nonheme Fe(II) site of homogentisate dioxygenase

Homogentisate 1,2-dioxygenase (HGDO) uses a mononuclear nonheme Fe2+ to catalyze the oxidative ring cleavage in the degradation of Tyr and Phe by producing maleylacetoacetate from homogentisate (2,5-dihydroxyphenylacetate). Here, we report three crystal structures of HGDO, revealing five different steps in its reaction cycle at 1.7–1.98 Å resolution. The resting state structure displays an octahedral coordination for Fe2+ with two histidine residues (His331 and His367), a bidentate carboxylate ligand (Glu337), and two water molecules. Homogentisate binds as a monodentate ligand to Fe2+, and its interaction with Tyr346 invokes the folding of a loop over the active site, effectively shielding it from solvent. Binding of homogentisate is driven by enthalpy and is entropically disfavored as shown by anoxic isothermal titration calorimetry. Three different reaction cycle intermediates have been trapped in different HGDO subunits of a single crystal showing the influence of crystal packing interactions on the course of enzymatic reactions. The observed superoxo:semiquinone-, alkylperoxo-, and product-bound intermediates have been resolved in a crystal grown anoxically with homogentisate, which was subsequently incubated with dioxygen. We demonstrate that, despite different folds, active site architectures, and Fe2+ coordination, extradiol dioxygenases can proceed through the same principal reaction intermediates to catalyze the O2-dependent cleavage of aromatic rings. Thus, convergent evolution of nonhomologous enzymes using the 2-His-1-carboxylate facial triad motif developed different solutions to stabilize closely related intermediates in unlike environments.

CooC1 from Carboxydothermus hydrogenoformans Is a Nickel-Binding ATPase

The maturation of nickel-dependent enzymes requires the participation of several accessory proteins. Typically the hydrolysis of nucleotides is necessary for the final metal transfer steps. The ATPase CooC has been implicated in the insertion of nickel into the Ni,Fe cluster (C cluster) of the carbon monoxide dehydrogenase from Rhodospirillum rubrum. Analysis of the amino acid sequence of CooC suggests the presence of motifs typical for the MinD family of SIMIBI class NTPases, which contain a deviant Walker A motif. The genome of the carboxidotrophic hydrogenogenic bacterium Carboxydothermus hydrogenoformans contains three open reading frames with distinct sequence homology to CooC from R. rubrum. We overproduced, isolated, and studied CooC1 from C. hydrogenoformans. As-isolated CooC1 is monomeric in the absence of ligands but dimerizes in the presence of either nickel, ADP, or ATP. CooC1 shows ATPase activity, and the ADP- and ATP-bound dimeric states are distinguished by their stability. The K8A mutant of CooC1, in which alanine replaces the signature lysine typical for the deviant Walker A motif in the MinD family, is incapable of both ATP hydrolysis and ATP-dependent dimerization. This corroborates that CooC1 is indeed a member of the MinD family and suggests an analogous dynamic equilibrium between monomeric and dimeric states. CooC proteins are involved in the insertion of nickel into carbon monoxide dehydrogenases, and we found that one CooC1 dimer binds one Ni(II) ion with nanomolar affinity. Ni-induced dimerization and the Ni(II)-CooC1 stoichiometry suggest that the Ni-binding site of CooC1 occurs in the dimer interface.

Crystal Structure of the ATP-Dependent Maturation Factor of Ni,Fe-Containing Carbon Monoxide Dehydrogenases

CooC proteins are ATPases involved in the incorporation of nickel into the complex active site ([Ni-4Fe-4S]) cluster of Ni,Fe-dependent carbon monoxide dehydrogenases. The genome of the carboxydotrophic bacterium Carboxydothermus hydrogenoformans encodes five carbon monoxide dehydrogenases and three CooC-type proteins, of which CooC1 was shown to be a nickel-binding ATPase. We determined the crystal structure of CooC1 in four different states: empty, ADP-bound, Zn(2+)/ADP-bound, and Zn(2+)-bound. The structure of CooC1 consists of two spatially separated functional modules: an ATPase module containing the deviant Walker A motif and a metal-binding module that confers the specific function of CooC1. The ATPase module is homologous to other members of the MinD family and, in analogy to the dimeric structure of ATP-bound Soj, is likely responsible for the ATP-dependent dimerization of CooC1. Its core topology classifies CooC1 as a member of the MinD family of SIMIBI (signal recognition particle, MinD and BioD)-class NTPases. The crystal structure of Zn(2+)-bound CooC1 reveals a conserved C-X-C motif as the metal-binding site responsible for metal-induced dimerization. The competitive binding of Ni(2+) and Zn(2+) to CooC1 in solution confirms that the conserved C-X-C motif is also responsible for the interaction with Ni(2+). A comparison of the different CooC1 structures determined suggests a mutual dependence of metal-binding site and nucleotide-binding site.

Function and transcriptional regulation of the isocitrate lyase in Pseudomonas aeruginosa

Pseudomonas aeruginosa ATCC 17933 is capable of growing aerobically on ethanol as sole source of carbon and energy. This requires the glyoxylate cycle for replenishing C4-compounds to the TCA cycle. The enzyme isocitrate lyase (ICL) catalyzes the first step of this glyoxylate shunt. Its activity was induced more than 10-fold in response to the carbon sources ethanol or acetate instead of glucose or succinate. We could prove that in P. aeruginosa ICL is essential for aerobic as well as anaerobic utilization of C2-sources. Transcriptional regulation of icl gene (aceA) expression was monitored on different carbon sources by using an aceA–lacZ gene fusion. A strong correlation between promoter and ICL activity indicated regulation at the transcriptional level. But ICL was not simply induced by the mere presence of ethanol in the growth medium as was demonstrated by cultivation on mixed substrates. P. aeruginosa showed diauxic growth on media containing ethanol–succinate or ethanol–glucose mixtures and did not transcribe the aceA gene to metabolize ethanol until succinate or glucose, respectively, were exhausted. Inactivation of the chromosomal aceA gene in P. aeruginosa led to an inability to grow on ethanol and acetate. Promoter activity studies showed that all genes necessary to oxidize ethanol were downregulated in the ICL-negative mutant. But on mixed substrates like ethanol–succinate or ethanol–glucose the mutant exhibited growth and utilized ethanol as well, probably as energy source only.

Redox-dependent complex formation by an ATP-dependent activator of the corrinoid/iron-sulfur protein

Movement, cell division, protein biosynthesis, electron transfer against an electrochemical gradient, and many more processes depend on energy conversions coupled to the hydrolysis of ATP. The reduction of metal sites with low reduction potentials (E0′ < -500 mV) is possible by connecting an energetical uphill electron transfer with the hydrolysis of ATP. The corrinoid-iron/sulfur protein (CoFeSP) operates within the reductive acetyl-CoA pathway by transferring a methyl group from methyltetrahydrofolate bound to a methyltransferase to the [Ni-Ni-Fe4S4] cluster of acetyl-CoA synthase. Methylation of CoFeSP only occurs in the low-potential Co(I) state, which can be sporadically oxidized to the inactive Co(II) state, making its reductive reactivation necessary. Here we show that an open-reading frame proximal to the structural genes of CoFeSP encodes an ATP-dependent reductive activator of CoFeSP. Our biochemical and structural analysis uncovers a unique type of reductive activator distinct from the electron-transferring ATPases found to reduce the MoFe-nitrogenase and 2-hydroxyacyl-CoA dehydratases. The CoFeSP activator contains an ASKHA domain (acetate and sugar kinases, Hsp70, and actin) harboring the ATP-binding site, which is also present in the activator of 2-hydroxyacyl-CoA dehydratases and a ferredoxin-like [2Fe-2S] cluster domain acting as electron donor. Complex formation between CoFeSP and its activator depends on the oxidation state of CoFeSP, which provides evidence for a unique strategy to achieve unidirectional electron transfer between two redox proteins.

Carbon Monoxide. Toxic Gas and Fuel for Anaerobes and Aerobes: Carbon Monoxide Dehydrogenases

Carbon monoxide (CO) pollutes the atmosphere and is toxic for respiring organisms including man. But CO is also an energy and carbon source for phylogenetically diverse microbes living under aerobic and anaerobic conditions. Use of CO as metabolic fuel for microbes relies on enzymes like carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS), which catalyze conversions resembling processes that eventually initiated the dawn of life.CODHs catalyze the (reversible) oxidation of CO with water to CO2 and come in two different flavors with unprecedented active site architectures. Aerobic bacteria employ a Cu- and Mo-containing CODH in which Cu activates CO and Mo activates water and takes up the two electrons generated in the reaction. Anaerobic bacteria and archaea use a Ni- and Fe-containing CODH, where Ni activates CO and Fe provides the nucleophilic water. Ni- and Fe-containing CODHs are frequently associated with ACS, where the CODH component reduces CO2 to CO and ACS condenses CO with a methyl group and CoA to acetyl-CoA.Our current state of knowledge on how the three enzymes catalyze these reactions will be summarized and the different strategies of CODHs to achieve the same task within different active site architectures compared.

Structural Basis for Electron and Methyl-Group Transfer in a Methyltransferase System Operating in the Reductive Acetyl-Coa Pathway

Several anaerobic acetogenic, methanogenic, hydrogenogenic, and sulfate-reducing microorganisms are able to use the reductive acetyl-CoA (Wood-Ljungdahl) pathway to convert CO₂ into biomass. The reductive acetyl-CoA pathway consists of two branches connected by the Co/Fe-containing corrinoid iron-sulfur protein (CoFeSP), which transfers a methyl group from a methyltransferase (MeTr)/methyltetrahydrofolate (CH₃-H₄ folate) complex to the reduced Ni-Ni-[4Fe-4S] cluster (cluster A) of acetyl-CoA synthase. We investigated the CoFeSP and MeTr couple of the hydrogenogenic bacterium Carboxydothermus hydrogenoformans and show that the two proteins are able to catalyze the methyl-group transfer reaction from CH₃-H₄ folate to the Co(I) center of CoFeSP. We determined the crystal structures of both proteins. The structure of CoFeSP includes the previously unresolved N-terminal domain of the large subunit of CoFeSP, revealing a unique four-helix-bundle-like architecture in which a [4Fe-4S] cluster is shielded by hydrophobic amino acids. It further reveals that the corrinoid and the [4Fe-4S] cluster binding domains are mobile, which is mandatory for the postulated electron transfer between them. Furthermore, we solved the crystal structures of apo-MeTr, CH₃-H₄-folate-bound MeTr, and H₄-folate-bound MeTr, revealing a substrate-induced closure of the CH₃-H₄ folate binding cavity of MeTr. We observed three different conformations of Asn200 depending on the substrate bound in the active site, demonstrating its conformational modulation by hydrogen-bonding interactions with the substrate. The observed flexibility could be essential to stabilize the transition state during methyl-group transfer. The conformational space and role of Asn200 are likely conserved in homologous cobalamin-dependent MeTrs such as methionine synthase.