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Unexpected NO-dependent DNA binding by the CooA homolog from Carboxydothermus hydrogenoformans.

ABSTRACT: CooA, the CO-sensing heme protein from Rhodospirillum rubrum, regulates the expression of genes that encode a CO-oxidation system, allowing R. rubrum to use CO as a sole energy source. To better understand the gas-sensing regulation mechanism used by R. rubrum CooA and its homologs in other organisms, we characterized spectroscopically and functionally the Fe(II), Fe(II)-NO, and Fe(II)-CO forms of CooA from Carboxydothermus hydrogenoformans. Surprisingly, and unlike R. rubrum CooA, C. hydrogenoformans CooA binds NO to form a six-coordinate Fe(II)-NO heme that is active for DNA binding in vitro and in vivo. In contrast, R. rubrum CooA, which is exquisitely specific for CO, forms a five-coordinate Fe(II)-NO adduct that is inactive for DNA binding. Based on analyses of protein variants and temperature studies, NO-dependent DNA binding by C. hydrogenoformans CooA is proposed to result from a greater apparent stability of the six-coordinate Fe(II)-NO adduct at room temperature. Results from the present study strengthen the proposal that CO specificity in the CooA activation mechanism is based on the requirement for a small, neutral distal ligand, which in turn affects the relative positioning of the ligand-bound heme.

SUBMITTER: Clark RW

PROVIDER: S-EPMC1347970 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Growth profile of Carboxydothermus hydrogenoformans on pyruvate.

Project description:Carboxydothermus hydrogenoformans is a thermophilic anaerobic strain most widely known for its ability to produce hydrogen (H2) when grown on carbon monoxide (CO). Although relatively well studied, growth characterization on pyruvate has never been assessed. The present work fully characterizes growth of the bacterium on pyruvate as a sole carbon source. C. hydrogenoformans demonstrated a growth rate of 0.03 h-1, with pyruvate consumption ranging between 0.21 and 0.48 mol?·?g-1 volatile suspended solid?·?d-1. A lag phase was also observed when switching from pyruvate to CO. When grown simultaneously on pyruvate and CO, pyruvate consumption was initiated upon CO depletion. This was attributed to pyruvate oxidation inhibition by CO, and not to a diauxic phenomenom. The strain also showed homoacetogenic activity.

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Project description:Carbon monoxide utilizing bacterium

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Novel physiological features of Carboxydothermus hydrogenoformans and Thermoterrabacterium ferrireducens.

Project description:Carboxydothermus hydrogenoformans is able to grow by conversion of CO to H2 and CO2. Besides CO, only pyruvate was described as serving as an energy source. Based on 16S rRNA gene sequence similarity, C. hydrogenoformans is closely related to Thermoterrabacterium ferrireducens. T. ferrireducens is like C. hydrogenoformans a gram-positive, thermophilic, strict anaerobic bacterium. However, it is capable of using various electron donors and acceptors for growth. Growth of C. hydrogenoformans with multiple electron donors and acceptors was tested. C. hydrogenoformans oxidized formate, lactate, glycerol, CO, and H2 with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor. Sulfite, thiosulfate, sulfur, nitrate, and fumarate were reduced with lactate as an electron donor. T. ferrireducens oxidized CO with 9,10-anthraquinone-2,6-disulfonate as an electron acceptor but did not produce H2 from CO. In contrast to what was published before, T. ferrireducens was able to grow on lactate with sulfite, sulfur, and nitrate as electron acceptors.

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Life in hot carbon monoxide: the complete genome sequence of Carboxydothermus hydrogenoformans Z-2901.

Project description:We report here the sequencing and analysis of the genome of the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. This species is a model for studies of hydrogenogens, which are diverse bacteria and archaea that grow anaerobically utilizing carbon monoxide (CO) as their sole carbon source and water as an electron acceptor, producing carbon dioxide and hydrogen as waste products. Organisms that make use of CO do so through carbon monoxide dehydrogenase complexes. Remarkably, analysis of the genome of C. hydrogenoformans reveals the presence of at least five highly differentiated anaerobic carbon monoxide dehydrogenase complexes, which may in part explain how this species is able to grow so much more rapidly on CO than many other species. Analysis of the genome also has provided many general insights into the metabolism of this organism which should make it easier to use it as a source of biologically produced hydrogen gas. One surprising finding is the presence of many genes previously found only in sporulating species in the Firmicutes Phylum. Although this species is also a Firmicutes, it was not known to sporulate previously. Here we show that it does sporulate and because it is missing many of the genes involved in sporulation in other species, this organism may serve as a "minimal" model for sporulation studies. In addition, using phylogenetic profile analysis, we have identified many uncharacterized gene families found in all known sporulating Firmicutes, but not in any non-sporulating bacteria, including a sigma factor not known to be involved in sporulation previously.

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Carboxydothermus hydrogenoformans Z-2901

Project description:Hyperthermophilic bacterium that can grow on carbon monoxide

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A functional Ni-Ni-[4Fe-4S] cluster in the monomeric acetyl-CoA synthase from Carboxydothermus hydrogenoformans.

Project description:In anaerobic microorganisms employing the acetyl-CoA pathway, acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH) form a complex (ACS/CODH) that catalyzes the synthesis of acetyl-CoA from CO, a methyl group, and CoA. Previously, a [4Fe-4S] cubane bridged to a copper-nickel binuclear site (active site cluster A of the ACS component) was identified in the ACS(Mt)/CODH(Mt) from Moorella thermoacetica whereas another study revealed a nickel-nickel site in the open form of ACS(Mt), and a zink-nickel site in the closed form. The ACS(Ch) of the hydrogenogenic bacterium Carboxydothermus hydrogenoformans was found to exist as an 82.2-kDa monomer as well as in a 1:1 molar complex with the 73.3-kDa CODHIII(Ch). Homogeneous ACS(Ch) and ACS(Ch)/CODHIII(Ch) catalyzed the exchange between [1-(14)C]acetyl-CoA and (12)CO with specific activities of 2.4 or 5.9 micromol of CO per min per mg, respectively, at 70 degrees C and pH 6.0. They also catalyzed the synthesis of acetyl-CoA from CO, methylcobalamin, corrinoid iron-sulfur protein, and CoA with specific activities of 0.14 or 0.91 micromol of acetyl-CoA formed per min per mg, respectively, at 70 degrees C and pH 7.3. The functional cluster A of ACS(Ch) contains a Ni-Ni-[4Fe-4S] site, in which the positions proximal and distal to the cubane are occupied by Ni ions. This result is apparent from a positive correlation of the Ni contents and negative correlations of the Cu or Zn contents with the acetyl-CoA/CO exchange activities of different preparations of monomeric ACS(Ch), a 2.2-A crystal structure of the dithionite-reduced monomer in an open conformation, and x-ray absorption spectroscopy.

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A 'Split-Gene' Transketolase From the Hyper-Thermophilic Bacterium <i>Carboxydothermus hydrogenoformans</i>: Structure and Biochemical Characterization.

Project description:A novel transketolase has been reconstituted from two separate polypeptide chains encoded by a 'split-gene' identified in the genome of the hyperthermophilic bacterium, Carboxydothermus hydrogenoformans. The reconstituted active α2β2 tetrameric enzyme has been biochemically characterized and its activity has been determined using a range of aldehydes including glycolaldehyde, phenylacetaldehyde and cyclohexanecarboxaldehyde as the ketol acceptor and hydroxypyruvate as the donor. This reaction proceeds to near 100% completion due to the release of the product carbon dioxide and can be used for the synthesis of a range of sugars of interest to the pharmaceutical industry. This novel reconstituted transketolase is thermally stable with no loss of activity after incubation for 1 h at 70°C and is stable after 1 h incubation with 50% of the organic solvents methanol, ethanol, isopropanol, DMSO, acetonitrile and acetone. The X-ray structure of the holo reconstituted α2β2 tetrameric transketolase has been determined to 1.4 Å resolution. In addition, the structure of an inactive tetrameric β4 protein has been determined to 1.9 Å resolution. The structure of the active reconstituted α2β2 enzyme has been compared to the structures of related enzymes; the E1 component of the pyruvate dehydrogenase complex and D-xylulose-5-phosphate synthase, in an attempt to rationalize differences in structure and substrate specificity between these enzymes. This is the first example of a reconstituted 'split-gene' transketolase to be biochemically and structurally characterized allowing its potential for industrial biocatalysis to be evaluated.

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Carboxydothermus hydrogenoformans strain:DSM 6008

Project description:Carboxydothermus hydrogenoformans strain:DSM 6008 Genome sequencing

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Effect of DNA binding on geminate CO recombination kinetics in CO-sensing transcription factor CooA.

Project description:Carbon monoxide oxidation activator (CooA) proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding in two CooA homologues, Rhodospirillum rubrum (RrCooA) and Carboxydothermus hydrogenoformans (ChCooA). The effects of DNA binding and the truncation of the DNA-binding domain on the CO geminate recombination kinetics were specifically investigated. The CO rebinding kinetics in these CooA complexes take place on ultrafast time scales but remain non-exponential over many decades in time. We show that this non-exponential kinetic response is due to a quenched enthalpic barrier distribution resulting from a distribution of heme geometries that is frozen or slowly evolving on the time scale of CO rebinding. We also show that, upon CO binding, the distal pocket of the heme in the CooA proteins relaxes to form a very efficient hydrophobic trap for CO. DNA binding further tightens the narrow distal pocket and slightly weakens the iron-proximal histidine bond. Comparison of the CO rebinding kinetics of RrCooA, truncated RrCooA, and DNA-bound RrCooA proteins reveals that the uncomplexed and inherently flexible DNA-binding domain adds additional structural heterogeneity to the heme doming coordinate. When CooA forms a complex with DNA, the flexibility of the DNA-binding domain decreases, and the distribution of the conformations available in the heme domain becomes restricted. The kinetic studies also offer insights into how the architecture of the heme environment can tune entropic barriers in order to control the geminate recombination of CO in heme proteins, whereas spin selection rules play a minor or non-existent role.

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