Marie Thérèse Giudici-Orticoni

[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.

A new iron-oxidizing/O2-reducing supercomplex spanning both inner and outer membranes, isolated from the extreme acidophile Acidithiobacillus ferrooxidans.

The iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans involves various metalloenzymes. Here we demonstrate that the oxygen reduction pathway from ferrous iron (named downhill pathway) is organized as a supercomplex constituted of proteins located in the outer and inner membranes as well as in the periplasm. For the first time, the outer membrane-bound cytochrome c Cyc2 was purified, and we showed that it is responsible for iron oxidation and determined that its redox potential is the highest measured to date for a cytochrome c. The organization of metalloproteins inside the supramolecular structure was specified by protein-protein interaction experiments. The isolated complex spanning the two membranes had iron oxidase as well as oxygen reductase activities, indicating functional electron transfer between the first iron electron acceptor, Cyc2, and the CuA center of cytochrome c oxidase aa3. This is the first characterization of a respirasome from an acidophilic bacterium. In Acidithiobacillus ferrooxidans,O2 reduction from ferrous iron must be coupled to the energy-consuming reduction of NAD+(P) from ferrous iron (uphill pathway) required for CO2 fixation and other anabolic processes. Besides the proteins involved in the O2 reduction, there were additional proteins in the supercomplex, involved in uphill pathway (bc complex and cytochrome Cyc42), suggesting a possible physical link between these two pathways.

Electrochemistry, AFM, and PM‐IRRA Spectroscopy of Immobilized Hydrogenase: Role of a Hydrophobic Helix in Enzyme Orientation for Efficient H2 Oxidation

Nickel–iron hydrogenase ([NiFe] Hase) catalyzes hydrogen splitting into protons and electrons, and is a potential biocatalyst in fuel cells. Three FeS clusters aligned as a conductive wire drive electrons from the [NiFe] active site to the surface of the enzyme, where the redox partner (including the electrode) binds. Direct enzyme connection gave access to thermodynamic and kinetic data of enzymatic reactions through direct electron transfer (DET). Mediated electron transfer (MET) allowed recreation of the physiological electron-transfer chain, and/or connection of unfavorably oriented enzymes. Previous work demonstrated that DET or MET processes for H2 oxidation by a soluble, O2-sensitive [NiFe] Hase from Desulfovibrio species could be controlled by electrostatic interaction. The presence of an acidic patch of amino acids, coupled to a dipole moment pointing towards the distal FeS cluster (positioned at the surface of the enzyme), allowed orientation of the enzyme, which turned upside down as a function of the charge on the electrochemical interface. Recently, we reported on the electrochemistry of membrane-bound Aquifex aeolicus (Aa) [NiFe] Hase, which exhibits outstanding resistance to O2, CO, and heat. [8–10] Efficient immobilization of this Hase was achieved on graphite electrodes, in aqueous electrolytes and ionic liquids, by encapsulation in carbon nanotube networks, or connection to a redox polymer. In contrast to the soluble, O2sensitive [NiFe] Hase, no specific orientation could be obtained by electrostatic interaction for Aa Hase, and thus control of the electron-transfer process was not possible. A model structure accordingly put forward a very different environment of the distal FeS cluster, with no charged amino acid patch, in accordance with the membrane anchorage. We analyze herein H2 oxidation by Aa Hase immobilized on self-assembled monolayers (SAMs) on gold electrodes as a function of both the length and the nature of the thiol derivative (see SI 1 and SI 2 in the Supporting Information). For the first time, AFM and polarization modulation infrared reflection adsorption (PM-IRRA) studies are reported for understanding Aa Hase orientation and its consequences for electron-transfer process in H2 oxidation. Positively charged 4-aminothiophenol (ArNH2) and negatively charged 6-mercaptohexanoic acid (C5COOH) SAMs both yield DET and MET processes for H2 oxidation (Figure 1a and b), and neither process is favored over the other. A mixed process was similarly observed for H2 oxidation at charged short-chain alkanethiols, which are known to bemore disordered. This strongly suggests that electroenzymatic H2 oxidation is linked to multiple orientations of Hase on top of the charged SAMs, and not to Hase present inside possible SAM defects. The lipophilic methylene blue (MB) molecule

Biocatalysts for fuel cells: efficient hydrogenase orientation for H2 oxidation at electrodes modified with carbon nanotubes

We report the modification of gold and graphite electrodes with commercially available carbon nanotubes for immobilization of Desulfovibrio fructosovorans [NiFe] hydrogenase, for hydrogen evolution or consumption. Multiwalled carbon nanotubes, single-walled carbon nanotubes (SWCNs), and amine-modified and carboxyl-functionalized SWCNs were used and compared throughout. Two separate methods were performed: covalent attachment of oriented hydrogenase by controlled architecture of carbon nanotubes at gold electrodes, and adsorption of hydrogenase at carbon-nanotube-coated pyrolytic graphite electrodes. In the case of self-assembled carbon nanotubes at gold electrodes, hydrogenase orientation based on electrostatic interaction with the electrode surface was found to control the electrocatalytic process for H2 oxidation. In the case of carbon nanotube coatings on pyrolytic graphite electrodes, catalysis was controlled more by the geometry of the nanotubes than by the orientation of the enzyme. Noticeably, shortened SWCNs were demonstrated to allow direct electron transfer and generate high and quite stable current densities for H2 oxidation via adsorbed hydrogenase, despite having many carboxylic surface functions that could yield unfavorable hydrogenase orientation for direct electron transfer. This result is attributable to the high degree of oxygenated surface functions in addition to the length of shortened SWCNs that yields highly divided materials.

An Unconventional Copper Protein Required for Cytochrome c Oxidase Respiratory Function under Extreme Acidic Conditions

Very little is known about the processes used by acidophile organisms to preserve stability and function of respiratory pathways. Here, we reveal a potential strategy of these organisms for protecting and keeping functional key enzymes under extreme conditions. Using Acidithiobacillus ferrooxidans, we have identified a protein belonging to a new cupredoxin subfamily, AcoP, for “acidophile CcO partner,” which is required for the cytochrome c oxidase (CcO) function. We show that it is a multifunctional copper protein with at least two roles as follows: (i) as a chaperone-like protein involved in the protection of the CuA center of the CcO complex and (ii) as a linker between the periplasmic cytochrome c and the inner membrane cytochrome c oxidase. It could represent an interesting model for investigating the multifunctionality of proteins known to be crucial in pathways of energy metabolism.

Rhodanese Functions as Sulfur Supplier for Key Enzymes in Sulfur Energy Metabolism

Background: The function of SbdP, a cytoplasmic rhodanese from Aquifex aeolicus, is unknown. Results: SbdP is involved in sulfur energy metabolism via its interaction with key redox enzymes. Conclusion: SbdP supplies long sulfur chains to enzyme-active sites. Significance: Rhodaneses are part of the substrate traffic in sulfur energy metabolism. How microorganisms obtain energy is a challenging topic, and there have been numerous studies on the mechanisms involved. Here, we focus on the energy substrate traffic in the hyperthermophilic bacterium Aquifex aeolicus. This bacterium can use insoluble sulfur as an energy substrate and has an intricate sulfur energy metabolism involving several sulfur-reducing and -oxidizing supercomplexes and enzymes. We demonstrate that the cytoplasmic rhodanese SbdP participates in this sulfur energy metabolism. Rhodaneses are a widespread family of proteins known to transfer sulfur atoms. We show that SbdP has also some unusual characteristics compared with other rhodaneses; it can load a long sulfur chain, and it can interact with more than one partner. Its partners (sulfur reductase and sulfur oxygenase reductase) are key enzymes of the sulfur energy metabolism of A. aeolicus and share the capacity to use long sulfur chains as substrate. We demonstrate a positive effect of SbdP, once loaded with sulfur chains, on sulfur reductase activity, most likely by optimizing substrate uptake. Taken together, these results lead us to propose a physiological role for SbdP as a carrier and sulfur chain donor to these key enzymes, therefore enabling channeling of sulfur substrate in the cell as well as greater efficiency of the sulfur energy metabolism of A. aeolicus.

Mechanism of Chloride Inhibition of Bilirubin Oxidases and Its Dependence on Potential and pH

Bilirubin oxidases (BODs) belong to the multi-copper oxidase (MCO) family and efficiently reduce O2 at neutral pH and in physiological conditions where chloride concentrations are over 100 mM. BODs were consequently considered to be Cl- resistant contrary to laccases. However, there has not been a detailed study on the related effect of chloride and pH on the redox state of immobilized BODs. Here, we investigate by electrochemistry the catalytic mechanism of O2 reduction by the thermostable Bacillus pumilus BOD immobilized on carbon nanofibers in the presence of NaCl. The addition of chloride results in the formation of a redox state of the enzyme, previously observed for different BODs and laccases, which is only active after a reductive step. This behavior has not been previously investigated. We show for the first time that the kinetics of formation of this state is strongly dependent on pH, temperature, Cl- concentration and on the applied redox potential. UV-visible spectroscopy allows us to correlate the inhibition process by chloride with the formation of the alternative resting form of the enzyme. We demonstrate that O2 is not required for its formation and show that the application of an oxidative potential is sufficient. In addition, our results suggest that the reactivation may proceed thought the T3 β.

Characterization of a new periplasmic single-domain rhodanese encoded by a sulfur-regulated gene in a hyperthermophilic bacterium Aquifex aeolicus

Rhodaneses (thiosulfate cyanide sulfurtransferases) are enzymes involved in the production of the sulfur in sulfane form, which has been suggested to be the relevant biologically active sulfur species. Rhodanese domains occur in the three major domains of life. We have characterized a new periplasmic single-domain rhodanese from a hyperthermophile bacterium, Aquifex aeolicus, with thiosulfate:cyanide transferase activity, Aq-1599. The oligomeric organization of the enzyme is stabilized by a disulfide bridge. To date this is the first characterization from a hyperthermophilic bacterium of a periplasmic sulfurtransferase with a disulfide bridge. The aq-1599 gene belongs to an operon that also contains a gene for a prepilin peptidase and that is up-regulated when sulfur is used as electron acceptor. Finally, we have observed a sulfur-dependent bacterial adherence linked to an absence of flagellin suggesting a possible role for sulfur detection by A. aeolicus.

Biomass for Energy: Energetic and Environmental Challenges of Biofuels

Transportation is 94 % dependent on oil, represents around 20 % of global consumption of energy, and is responsible for 23 % of total emissions from fossil fuels. For several years, progress has been made to enhance the energy efficiency of the systems, but increasing the part of biofuel still seems irremediable both for environmental, economic, and energy independence reasons. Fuel production from biomass is clearly considered as an important substitute for liquid fossil fuels such as bioethanol for motor gasoline, biodiesel for diesel, jet fuel for biokerosene, and for gaseous fuels (hydrogen, natural gas for vehicles, biomethane, etc.). This chapter presents the main pathways for the production of biofuels, and classifies their degree of maturity: The first-generation processes that value the reserves of a plant (starch, sugar, oil) are now mature and industrially deployed. The second generation processes extend their resource to the whole plant tissues (agricultural, forest) or to organic waste, and are almost under scientific control but they still need more economic and energetic assessment before being commercially deployed. The last innovative pathway, the advanced or third biofuel generation, shows significant potential by using bioalgae or microorganisms capable of producing much more biomass oil convertible into biodiesel and gaseous fuels such as methane or hydrogen.

A New Acid-Stable, Fe-Oxidizing/O2-Reducing Supercomplex Spanning Both Inner and Outer Membranes, Isolated from the Extremophile Acidithiobacillus

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic gram-negative bacterium that derives energy from the oxidation of Fe(II) ions, elemental sulfur and various sulfur compounds at pH 2 using oxygen as electron acceptor. This organism is the main bacterium used in the industrial extraction of copper and uranium from ores using the microbial leaching technique. The study of this bacterium presents economic and fundamental biological interests. It oxidizes metal sulfides like pyrite, thereby enabling solubilisation and valorization of precious metals. Acidithiobacillus contains various soluble (cytochromes c4, rusticyanin) and membrane-bound (aa3-type cytochrome c oxidase, cytochrome Cyc2) electron carriers suspected to be involved in electron transfer from Fe(II) ions to molecular oxygen. We have shown, after membrane solubilisation, purification and proteomics analysis, that proteins encoded by the rus operon are associated in a macromolecular complex in this bacterium: the outer membrane-bound cytochrome c Cyc2, the periplasmic soluble cytochrome c4 Cyc1 and rusticyanin, the inner membrane-bound cytochrome c oxidase and an hypothetical membrane-bound protein ORF1 which function is still unknown. This complex contains also proteins from the bc complex (encoded by the petI operon) and a major outer membrane protein OMP40. Sequences analysis has shown that ORF1 might be a chaperon for cytochrome c oxidase maturation and Cu fixation. EPR analysis revealed that this protein probably contains Cu. The multiprotein complex is functionally active as measured by kinetics experiments using Fe(II) ions as electron donor. This is the first characterization of a complex containing soluble and membranous proteins from outer and inner membrane from an acidophilic bacterium. This association in a supercomplex allows a direct electron transfer from Fe(II) ions to cytochrome oxidase to minimize energetic losses. Advanced Materials Research Online: 2007-07-15 ISSN: 1662-8985, Vols. 20-21, pp 572-572 doi:10.4028/www.scientific.net/AMR.20-21.572