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.

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(-).

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. 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.

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 (E 0 0 < −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-Fe 4 S 4 ] cluster of acetyl-CoA synthase. Methylation of CoFeSP only occurs in the lowpotential 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.

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. The Ni(2+) ion is coordinated by three histidine residues and a glutamate residue (E(76)) in all three states. Upon binding, quercetin replaces one water ligand at Ni and is stabilized by a short hydrogen bond through E(76) , the carboxylate group of which rotates by 90°. This conformational change weakens the interaction between Ni and the remaining water ligand, thereby preparing a coordination site at Ni to bind O2. O2 binds side-on to the Ni(2+) ion and is perpendicular to the C2-C3 and C3-C4 bonds of quercetin, which are cleaved in the following reaction steps.

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.

Homogentisate 1,2-dioxygenase (HGDO) uses a mononuclear nonheme Fe 2+ 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 Fe 2+ with two histidine residues (His331 and His367), a bidentate carboxylate ligand (Glu337), and two water molecules. Homogentisate binds as a monodentate ligand to Fe 2+, 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 Fe 2+ coordination, extradiol dioxygenases can proceed through the same principal reaction intermediates to catalyze the O 2 -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.dioxygen activation | non-heme iron | amino acid degradation | Pseudomonas putida | alkaptonuria H omogentisate (2,5-dihydroxyphenylacetate, HG) is the central metabolite in the degradation pathways of phenylalanine and tyrosine in aerobic organisms ranging from soil bacteria like Pseudomonas putida to man (1, 2). Oxidative ring cleavage is catalyzed by homogentisate 1,2-dioxygenase (HGDO), which incorporates the atoms of molecular oxygen into HG to produce maleylacetoactate (1, 3-6). A deficiency of HGDO is known to cause the autosomal recessive disorder alkaptonuria in humans, the first genetic defect to be recognized as such (6-8). The crystal structure of HGDO from man (HGDO Hs ) revealed that the enzyme belongs to the cupin fold type of nonheme iron-dependent dioxygenases, forming a homohexamer consisting of a dimer of trimers (6). The active site of HGDO Hs employs the 2-His-1-carboxylate facial triad to bind an essential Fe 2+ ion with a distorted square-pyramidal arrangement (6). The facial triad motif is found in various nonhomologous types of nonheme Fe 2+ -dependent enzymes and commonly serves to control the reactivity of the Fe 2+ site and to activate an aromatic or aliphatic substrate together with dioxygen (9) and is also featured in extradiol-type dioxygenases such as 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) and homoprotocatechuate 2,3...

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