Mireille Bruschi

Enzymatic reduction of chromate: comparative studies using sulfate-reducing bacteria

Abstract Various sulfate-reducing bacteria of the genera Desulfovibrio and Desulfomicrobium were tested and compared for enzymatic reduction of chromate. Our study demonstrated that the ability to reduce chromate is widespread among sulfate-reducing bacteria. Among them, Desulfomicrobium norvegicum reduced Cr(VI) with the highest reaction rate. This strain grew in the presence of up to 500 μM chromate, but Cr(VI) reduction in the absence of sulfate was not associated with growth. The presence of chromate induced morphological changes and leakage of periplasmic proteins into the medium. The ability of isolated polyheme cytochromes c from sulfate- and sulfur-reducing bacteria to reduce chromate was also analyzed. Tetraheme cytochrome c3(Mr. 13,000) from Desulfomicrobium norvegicum showed twice as much activity as either tetraheme cytochrome c3 from Desulfovibrio vulgaris strain Hildenborough or triheme cytochrome c7 from Desulfuromonas acetoxidans. Results with cytochromes c3 and other c-type cytochromes altered by site-directed mutagenesis indicated that negative redox potential hemes are crucial for metal reductase activity. The present study also demonstrated that the (Fe) hydrogenase from sulfate-reducing bacteria could reduce chromate.

Characterization of the periplasmic hydrogenase from Desulfovibrio gigas

Abstract The hydrogenase of the sulfate-reducer Desulfovibrio gigas has been purified to homogeneity. The pure enzyme shows a specific activity of 90 μmoles H2 evolved/min./mg protein. Its molecular weight is 89,500 and its is composed of two different subunits (mol. wt. : 62,000 and 26,000) which are not covalently bound. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein. The millimolar extinction coefficients of the hydrogenase are 46.5 and 170 respectively at 400 and 280 nm. It contains about 12 iron atoms and 12 acid-labile sulfur groups per molecule and the quantitative extrusion of the Fe-S centers of the hydrogenase indicates the presence of 3 Fe4S4 clusters. This hydrogenase has 21 half-cystine residues per molecule and a preponderance of aromatic amino-acids.

[NiFe] hydrogenases from the hyperthermophilic bacterium Aquifex aeolicus : properties, function, and phylogenetics

Genes potentially coding for three distinct [NiFe] hydrogenases are present in the genome of Aquifex aeolicus. We have demonstrated that all three hydrogenases are expressed under standard growth conditions of the organism. Two hydrogenases were further purified to homogeneity. A periplasmically oriented hydrogenase was obtained in two forms, i.e., as a soluble enzyme containing only the two essential subunits and as a detergent-solubilized complex additionally containing a membrane-integral b-type cytochrome. The second hydrogenase purified was identified as a soluble cytoplasmic enzyme. The isolated enzymes were characterized with respect to biochemical/biophysical parameters, activity, thermostability, and substrate specificity. The phylogenetic positioning of all three hydrogenases was analyzed. A model for the metabolic roles of the three enzymes is proposed on the basis of the obtained results.

Adaptation to extreme environments: macromolecular dynamics in bacteria compared in vivo by neutron scattering

Mean macromolecular dynamics was quantified in vivo by neutron scattering in psychrophile, mesophile, thermophile and hyperthermophile bacteria. Root mean square atomic fluctuation amplitudes determining macromolecular flexibility were found to be similar for each organism at its physiological temperature (∼1 Å in the 0.1 ns timescale). Effective force constants determining the mean macromolecular resilience were found to increase with physiological temperature from 0.2 N/m for the psychrophiles, which grow at 4°C, to 0.6 N/m for the hyperthermophiles (85°C), indicating that the increase in stabilization free energy is dominated by enthalpic rather than entropic terms. Larger resilience allows macromolecular stability at high temperatures, while maintaining flexibility within acceptable limits for biological activity.

Rubredoxin from Desulfovibrio gigas. A molecular model of the oxidized form at 1.4 A resolution.

The crystal structure of rubredoxin from the sulfate-reducing bacterium Desulfovibrio gigas has been determined at 1.4 A resolution (1 A = 0.1 nm) by X-ray diffraction methods; starting with a model of the isostructural rubredoxin from Desulfovibrio vulgaris. Refinement of the molecular model has been carried out by restrained least-squares techniques and Fourier series calculations. The present model includes a formyl at the N-terminal end and 121 possible sites for solvent molecules with full or partial occupancy, which corresponds to the modeling of nearly all the solvent medium. The crystallographic R factor against the data with 10 A greater than d greater than 1.4 A with F greater than 2 sig(F), is 0.136; and R = 0.140 when all the data are considered. The estimated average root-mean-square (r.m.s.) error on the positional parameters is about 0.12 A. The overall structural features of this molecule are close to those of the two highly refined rubredoxins from Clostridium pasteurianum and D. vulgaris. Superposition of these two molecules on the rubredoxin from D. gigas shows in both cases an overall r.m.s. deviation of 0.5 A for the atoms in the main-chain and of 0.4 A for the atoms in the side-chains that make up the hydrophobic core. The iron atom is co-ordinated to four cysteine sulfur atoms forming an almost regular tetrahedron, with Fe-SG distances ranging from 2.27 A to 2.31 A and angles varying from 103 degrees to 115 degrees. The intramolecular hydrogen-bonding pattern is quite comparable to those found in other proteins refined at high resolution. All the polar groups are involved in hydrogen bonds: intramolecular, intermolecular or with solvent molecules. The main structural differences from the other rubredoxins are in the nature and the distribution of some of the charged residues over the molecular surface. The possible influence of several structural factors on the intramolecular and intermolecular electron transfer properties such as the NH...SG bonds, the solvent exposure of the redox center, and the aromatic core is discussed. The conservation, during evolution, of a ring of acidic residues in the proximity of the FeSG4 center suggests that this ring may be implicated in the recognition processes between rubredoxins and their functional partners.

Hydrogenases in sulfate-reducing bacteria function as chromium reductase

AbstractThe ability of sulfate-reducing bacteria (SRB) to reduce chromate VI has been studied for possible application to the decontamination of polluted environments. Metal reduction can be achieved both chemically, by H2S produced by the bacteria, and enzymatically, by polyhemic cytochromes c3. We demonstrate that, in addition to low potential polyheme c-type cytochromes, the ability to reduce chromate is widespread among [Fe], [NiFe], and [NiFeSe] hydrogenases isolated from SRB of the genera Desulfovibrio and Desulfomicrobium. Among them, the [Fe] hydrogenase from Desulfovibrio vulgaris strain Hildenborough reduces Cr(VI) with the highest rate. Both [Fe] and [NiFeSe] enzymes exhibit the same Km towards Cr(VI), suggesting that Cr(VI) reduction rates are directly correlated with hydrogen consumption rates. Electron paramagnetic resonance spectroscopy enabled us to probe the oxidation by Cr(VI) of the various metal centers in both [NiFe] and [Fe] hydrogenases. These experiments showed that Cr(VI) is reduced to paramagnetic Cr(III), and revealed inhibition of the enzyme at high Cr(VI) concentrations. The significant decrease of both hydrogenase and Cr(VI)-reductase activities in a mutant lacking [Fe] hydrogenase demonstrated the involvement of this enzyme in Cr(VI) reduction in vivo. Experiments with [3Fe-4S] ferredoxin from Desulfovibrio gigas demonstrated that the low redox [Fe-S] (non-heme iron) clusters are involved in the mechanism of metal reduction by hydrogenases.

Bioremediation of chromate: thermodynamic analysis of the effects of Cr(VI) on sulfate-reducing bacteria.

Abstract. Developing new bioremediation processes for soils and effluents polluted by Cr(VI) requires the selection of the most efficient and the most heavy-metal-resistant bacteria. The effects of Cr(VI) on bioenergetic metabolism in two sulfate-reducing bacteria (SRB), Desulfovibrio vulgaris Hildenborough and Desulfomicrobium norvegicum, were monitored using isothermal microcalorimetry. The complete reduction of Cr(VI) to Cr(III) was studied by spectrophotometry and by speciation using a combination of high-performance liquid chromatography and inductively coupled plasma-mass spectrometry. Results revealed that Cr(VI) induces an inhibition of growth with concomitant production of energy, which can be compared to the reaction of the bacteria to a stress such as oxidative stress. Moreover, the sensitivity of bacteria towards this metal is as a characteristic of the strain, which leads to differences in the kinetics of Cr(VI) reduction. The study by microcalorimetry of heavy metal effects on SRB bioenergetic metabolism thus appears an appropriate tool to identify better strains to be used for industrial bioremediation process development.

Crystal structure of cytochrome c3 from Desulfovibrio desulfuricans Norway at 1.7 A resolution.

The crystal structure of cytochrome c3 (M(r) 13,000) from Desulfovibrio desulfuricans (118 residues, four heme groups) has been crystallographically refined to 1.7 A resolution using a simulated annealing method, based on the structure-model at 2.5 A resolution, already published. The final R-factor for 10,549 reflections was 0.198 covering the range from 5.5 to 1.7 A resolution. The individual temperature factors were refined for a total of 1059 protein atoms, together with 126 bound solvent molecules. The structure has been analyzed with respect to its detailed conformational properties, secondary structure features, temperature factor behaviour, bound solvent sites and heme geometry and ligation. The characteristic secondary structures of the polypeptide chain of this molecule are one extended alpha-helix, a short beta-strand and 13 reverse turns. The four heme groups are located in different structural environments, all highly exposed to solvent. The particular structural features of the heme environments are compared to the four hemes of the cytochrome c3 from Desulfovibrio vulgaris Miyazaki.

Correlations studies between structural and redox properties of cytochromes C3

Individual redox potential values are determined for different cytochromes c3. These polyhaemic cytochromes can be divided into two groups: one characterized by only one marked reduction step, the other one giving at least two well-marked reduction steps corresponding to redox potential values ranging from - 0.120 to - 0.400 V. Correlations between potential values and structural data are discussed.

Isolation and characterization of desulforedoxin, a new type of non-heme iron protein from Desulfovibrio gigas

Abstract Desulfovibrio gigas desulforedoxin is a new type of non-heme iron protein of molecular weight 7,900. It contains two iron atoms, no labile sulfide and eight cysteine residues per molecule. The optical spectrum of the oxidized form presents important differences from that of the rubredoxin type proteins. Upon reduction with dithionite there is no contribution to the visible region. The oxidation-reduction process is reversible but the protein is sensitive to repeated redox cycles. The protein contains 73 amino-acid residues. Like rubredoxin it does not contain histidine and arginine but it also lacks proline, isoleucine, phenylalanine, and tryptophan. The N-terminal sequence has been determined up to 35 residues and no evident homology was found with other non-heme iron proteins.