Biomedical subjects

Mireille Bruschi

Publications and source records attributed to Mireille Bruschi.

12 recordsLinked to original sources

PubMed2007

The bacterial metallome: composition and stability with specific reference to the anaerobic bacterium Desulfovibrio desulfuricans.

In bacteria, the intracellular metal content or metallome reflects the metabolic requirements of the cell. When comparing the composition of metals in phytoplankton and bacteria that make up the macronutrients and the trace elements, we have determined that the content of trace elements in both of these microorganisms is markedly similar. The trace metals consisting of transition metals plus zinc are present in a stoichometric molar formula that we have calculated to be as follows: Fe(1)Mn(0.3)Zn(0.26)Cu(0.03)Co(0.03)Mo(0.03). Under conditions of routine cultivation, trace metal homeostasis may be maintained by a series of transporter systems that are energized by the cell. In specific environments where heavy metals are present at toxic levels, some bacteria have developed a detoxification strategy where the metallic ion is reduced outside of the cell. The result of this extracellular metabolism is that the bacterial metallome specific for trace metals is not disrupted. One of the microorganisms that reduces toxic metals outside of the cell is the sulfate-reducing bacterium Desulfovibrio desulfuricans. While D. desulfuricans reduces metals by enzymatic processes involving polyhemic cytochromes c3 and hydrogenases, which are all present inside the cell; we report the presence of chain B cytochrome c nitrite reductase, NrfA, in the outer membrane fraction of D. desulfuricans ATCC 27774 and discuss its activity as a metal reductase.

Bacterial Proteins↗

PubMed2006

Cr(VI) quantification using an amperometric enzyme-based sensor: interference and physical and chemical factors controlling the biosensor response in ground waters.

The development of an amperometric enzyme-based sensor for chromate (CrO(4)(2-)) quantification in ground waters was investigated. Crucial physical and chemical factors characterising ground waters were tested for their influence or interference on chromate quantification: pH (7.6-8.5), temperature (9-25 degrees C), ionic strength (0-0.2M), oxygen, metals, bicarbonate and sulphate. The biosensor's response was dependent on temperature and pH as sensitivity increased with temperature and was higher at pH 7.6 than at pH 8.5. Sensitivity decreased with ionic strength until 0.1M, and was stable for higher values. Dissolved oxygen did not allow chromate quantification when it was present, but O(2) could be eliminated by adding Na(2)SO(3) or bubbling nitrogen gas into the solution. Bicarbonate did not interfere with chromate quantification by the biosensor. Sulphate was detected with a detection threshold 80 times higher than that of chromate and a lower sensitivity. Several metals (V(V), W(VI), Mn(VII), Mo(VI)) similar to chromate due to their oxidative properties and structure (oxyanions) were tested as possible interfering compounds. The sensitivity of the biosensor for these metals was low and the detection level was 30 times higher than that of chromate. These metal concentrations are usually weaker than chromate concentration in polluted ground waters so that dilution of the sample should allow chromate quantification by the biosensor. This study shows that the cytochrome c(3)-based sensor can detect compounds other than chromate but with a lower sensitivity. Although non-specific for the detection of chromate, it can however be adapted and used for the quantification of chromate in ground waters containing low sulphate concentration.

Anions↗

PubMed2005

Cr(VI) detoxification by Desulfovibrio vulgaris strain Hildenborough: microbe-metal interactions studies.

Toxic heavy metals constitute a worldwide environmental pollution problem. Bioremediation technologies represent efficient alternatives to the classic cleaning-up of contaminated soil and ground water. Most toxic heavy metals such as chromium are less soluble and toxic when reduced than when oxidized. Sulfate-reducing bacteria (SRB) are able to reduce heavy metals by a chemical reduction via the production of H2S and by a direct enzymatic process involving hydrogenases and c3 cytochromes. We have previously reported the effects of chromate [Cr(VI)] on SRB bioenergetic metabolism and the molecular mechanism of the metal reduction by polyhemic cytochromes. In the current work, we pinpoint the bacteria-metal interactions using Desulfovibrio vulgaris strain Hildenborough as a model. The bacteria were grown in the presence of high Cr(VI) concentration, where they accumulated precipitates of a reduced form of chromium, trivalent chromium [Cr(III)], on their cell surfaces. Moreover, the inner and outer membranes exhibited precipitates that shared the spectroscopic signature of trivalent chromium. This subcellular localization is consistent with enzymatic metal reduction by cytochromes and hydrogenases. Regarding environmental significance, our findings point out the Cr(VI) immobilization mechanisms of SRB; suggesting that SRB are highly important in metal biogeochemistry.

Biodegradation, Environmental↗

PubMed2005

Insight into molecular stability and physiological properties of the diheme cytochrome CYC41 from the acidophilic bacterium Acidithiobacillus ferrooxidans.

The cyc1 gene encoding the soluble dihemic cytochrome c CYC(41) from Acidithiobacillus ferrooxidans, an acidophilic organism, has been cloned and expressed in Escherichia coli as the host organism. The cytochrome was successfully produced and folded only in fermentative conditions: this allowed us to determine the molecular basis of the heme insertion at extreme pH. Point mutations at two sequence positions (E121 and Y63) were introduced near the two hemes in order to assign individual redox potentials to the hemes and to identify the interaction sites with the redox partners, rusticyanin and cytochrome oxidase. Characterization of mutants E121A, Y63A, and Y63F CYC(41) with biochemical and biophysical techniques were carried out. Substitution of tyrosine 63 by phenylalanine alters the environment of heme B. This result indicates that heme B has the lower redox potential. Interaction studies with the two physiological partners indicate that CYC(41) functions as an electron wire between RCy and cytochrome oxidase. A specific glutamate residue (E121) located near heme A is directly involved in the interaction with RCy. A docking analysis of CYC(41), RCy, and cytochrome oxidase allowed us to propose a model for the complex in agreement with our experimental data.

Acidithiobacillus↗

PubMed2005

Interaction and electron transfer between the high molecular weight cytochrome and cytochrome c3 from Desulfovibrio vulgaris Hildenborough: kinetic, microcalorimetric, EPR and electrochemical studies.

The complex formation between the tetraheme cytochrome c3 and hexadecaheme high molecular weight cytochrome c (Hmc), the structure of which has recently been resolved, has been characterized by cross-linking experiments, EPR, electrochemistry and kinetic analysis, and some key parameters of the interaction were determined. The analysis of electron transfer between [Fe] hydrogenase, cytochrome c3 and Hmc demonstrates a redox-shuttling role of cytochrome c3 in the pathway from hydrogenase to Hmc, and shows an effect of redox state on the interaction between the two cytochromes. The role of polyheme cytochromes in electron transfer from periplasmic hydrogenase to membrane redox proteins is assessed. A model with cytochrome c3 as an intermediate between hydrogenase and various polyheme cytochromes is proposed and its physiological consequences are discussed.

Bacterial Proteins↗

PubMed2004

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 ( approximately 1 A 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 degrees C, to 0.6 N/m for the hyperthermophiles (85 degrees 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.

Adaptation, Psychological↗

PubMed2003

Amperometric cytochrome c3-based biosensor for chromate determination.

The chromate reductase activity of cytochrome c(3) (Cyt c(3), M(r) 13000), isolated from the sulfate-reducing bacterium Desulfomicrobium norvegicum, was used to develop an amperometric biosensor to measure chromate (CrO(4)(2-)) bioavailability. The performance of various biosensor configurations for qualitative and quantitative determination of Cr(VI) was studied. Biosensor properties depend on the technique used to immobilize the enzyme on the electrode (glassy carbon electrode). Immobilization of Cyt c(3) by entrapment in poly 3,4-ethylenedioxythiophene films denatured the enzyme, while application of an adsorption technique did not affect enzyme activity but the detection range was limited. The best results were obtained with dialysis membranes, which allowed the determination of Cr(VI) from 0.20 to 6.84 mg l(-1) (3.85-132 microM) with a sensitivity of 35 nA mg(-1) l (1.82 nA microM(-1)). No interference was observed with As(V), As(III) and Fe(III). Only a small amount of Cyt c(3) (372 ng of protein) was needed for this biosensor.

Biosensing Techniques↗

PubMed2003

[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.

Amino Acid Sequence↗

PubMed2003

The structure of Acidithiobacillus ferrooxidans c(4)-cytochrome: a model for complex-induced electron transfer tuning.

The study of electron transfer between the copper protein rusticyanin (RCy) and the c(4)-cytochrome CYC(41) of the acidophilic bacterium Acidithiobacillus ferrooxidans has evidenced a remarkable decrease of RCy's redox potential upon complex formation. The structure of the CYC(41) obtained at 2.2 A resolution highlighted a specific glutamate residue (E121) involved in zinc binding as potentially playing a central role in this effect, required for the electron transfer to occur. EPR and stopped-flow experiments confirmed the strong inhibitory effect of divalent cations on CYC(41):RCy complex formation. A docking analysis of the CYC(41) and RCy structure allows us to propose a detailed model for the complex-induced tuning of electron transfer in agreement with our experimental data, which could be representative of other copper proteins involved in electron transfer.

Acidithiobacillus↗

PubMed2002

The metal reductase activity of some multiheme cytochromes c: NMR structural characterization of the reduction of chromium(VI) to chromium(III) by cytochrome c(7).

The redox reaction between CrO(4)(2-) and the fully reduced three-heme cytochrome c(7) from Desulfuromonas acetoxidans to give chromium(III) and the fully oxidized protein has been followed by NMR spectroscopy. The hyperfine coupling between the oxidized protein protons and chromium(III), which remains bound to the protein, gives rise to line-broadening effects on the NMR resonances that can be transformed into proton-metal distance restraints. Structure calculations based on these unconventional constraints allowed us to demonstrate that chromium(III) binds at a unique site and to locate it on the protein surface. The metal ion is located 7.9 +/- 0.4 A from the iron of heme IV, 16.3 +/- 0.7 A from the iron of heme III, and 22.5 +/- 0.5 A from the iron of heme I. Shift changes caused by the presence of unreactive MoO(4)(2-), a CrO(4)(2-) analogue, indicate the involvement of the same protein area in the anion binding. The titration of the oxidation of cytochrome c(7) shows a detailed mechanism of action. The presence of a specific binding site supports the hypothesis of the biological role of this cytochrome as a metal reductase.

Binding Sites↗

PubMed2002

The crystal structure of the hexadeca-heme cytochrome Hmc and a structural model of its complex with cytochrome c(3).

Sulfate-reducing bacteria contain a variety of multi-heme c-type cytochromes. The cytochrome of highest molecular weight (Hmc) contains 16 heme groups and is part of a transmembrane complex involved in the sulfate respiration pathway. We present the 2.42 A resolution crystal structure of the Desulfovibrio vulgaris Hildenborough cytochrome Hmc and a structural model of the complex with its physiological electron transfer partner, cytochrome c(3), obtained by NMR restrained soft-docking calculations. The Hmc is composed of three domains, which exist independently in different sulfate-reducing species, namely cytochrome c(3), cytochrome c(7), and Hcc. The complex involves the last heme at the C-terminal region of the V-shaped Hmc and heme 4 of cytochrome c(3), and represents an example for specific cytochrome-cytochrome interaction.

Crystallography, X-Ray↗

PubMed2002

A quick solution structure determination of the fully oxidized double mutant K9-10A cytochrome c7 from Desulfuromonas acetoxidans and mechanistic implications.

Lysines 9 and 10 in Desulfuromonas acetoxidans cytochrome c7, which could be involved in the interaction mechanism with the redox partners, have been replaced by alanine residues using site-directed mutagenesis. The solution structure of the fully oxidized form of K9-10A cytochrome c7, which is paramagnetic with three paramagnetic centers, has been determined via 1H NMR. The assignment of the spectra has been performed through an automatic program whose algorithm and strategy are here described. The assignment of the NOESY spectra has been further extended by back calculating the NOESY maps. The final number of meaningful NOE-based upper distance limits was 1186. In the Restrained Energy Minimization calculations, 147 pseudocontact shift constraints were also included, which showed consistency with NOE-based constraints and therefore further contribute to validate the structure quality. A final family of 35 conformers was calculated with RMSD values with respect to the mean structure of 0.69 +/- 0.17 A and 1.05 +/- 0.14 A for the backbone and heavy atoms, respectively. The overall fold of the molecule is maintained with respect to the native protein. The loop present between heme III and heme IV results to be highly disordered also in the present structure although its overall shape mainly resembles that of the oxidized native protein, and the two strands which give rise to the short beta-sheet present at the N-terminus and connected by a turn containing the mutated residues, are less clearly defined. If this loop is neglected, the RMSD values are 0.52 +/- 0.07 A and 0.92 +/- 0.06 A for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The relative distances and orientations of the three hemes are maintained, as well as the orientation of the imidazole rings of the axial histidine ligands, with the only exception of heme IV. Such difference probably reflects minor conformational changes due to the substitution of the vicinal Lys 10 with an Ala. The replacement of the two lysines does not affect the reduction potentials of the three hemes, consistently with the expectations on the basis of the structure and electrostatic calculations. However, the replacement of the two lysines affects the reactivity of the mutant cytochrome c7 with [Fe] hydrogenase, inducing a change in Km. This finding is in agreement with the identification of the protein area around heme IV as the interacting site.

Amino Acid Substitution↗