Marianne Guiral

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.

A Membrane-bound Multienzyme, Hydrogen-oxidizing, and Sulfur-reducing Complex from the Hyperthermophilic Bacterium Aquifex aeolicus

Aquifex aeolicus is a hyperthermophilic, chemolithoautotrophic, hydrogen-oxidizing, and microaerophilic bacterium growing at 85 °C. We have shown that it can grow on an H2/S° medium and produce H2S from sulfur in the later exponential phase. The complex carrying the sulfur reducing activity (electron transport from H2 to S°) has been purified and characterized. It is a membrane-bound multiprotein complex containing a [NiFe] hydrogenase and a sulfur reductase connected via quinones. The sulfur reductase is encoded by an operon annotated dms (dimethyl sulfoxide reductase) that we have renamed sre and is composed of three subunits. Sequence analysis showed that it belongs to the Me2SO reductase molybdoenzyme family and is similar to the sulfur/polysulfide/thiosulfate/tetrathionate reductases. The study of catalytic properties clearly demonstrated that it can reduce tetrathionate, sulfur, and polysulfide, but cannot reduce Me2SO and thiosulfate, and that NADPH increases the sulfur reducing activity. To date, this is the first characterization of a supercomplex from a bacterium that couples hydrogen oxidation and sulfur reduction. The distinctive feature in A. aeolicus is the cytoplasmic localization of the sulfur reduction, which is in accordance with the presence of sulfur globules in the cytoplasm. Association of this sulfur-reducing complex with a hydrogen-oxygen pathway complex (hydrogenase I, bc1 complex) in the membrane suggests that subcomplexes involved in respiratory chains in this bacterium are part of supramolecular organization.

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

TorT, a member of a new periplasmic binding protein family, triggers induction of the Tor respiratory system upon trimethylamine N-oxide electron-acceptor binding in Escherichia coli.

In anaerobiosis, Escherichia coli can use trimethylamine N-oxide (TMAO) as a terminal electron acceptor. Reduction of TMAO in trimethylamine (TMA) is mainly performed by the respiratory TMAO reductase. This system is encoded by the torCAD operon, which is induced in the presence of TMAO. This regulation involves a two-component system comprising TorS, an unorthodox histidine kinase, and TorR, a response regulator. A third protein, TorT, sharing homologies with periplasmic binding proteins, plays a key role in this regulation because disruption of the torT gene abolishes tor expression. In this study we showed that TMAO protects TorT against degradation by the GluC endoproteinase and modifies its temperature-induced CD spectrum. We also isolated a TorT negative mutant that is no longer protected by TMAO from degradation by GluC. Isothermal titration calorimetry confirmed that TorT binds TMAO with a binding constant of 150 μm. Therefore, we conclude that TorT binds TMAO and that this binding promotes a conformational change of TorT. We also showed that TorT interacts with the periplasmic domain of TorS in both the presence and absence of TMAO but the TorT-TMAO complex induces a higher GluC protection of TorS than TorT alone. These results support the idea that TMAO binding to TorT induces a cascade of conformational changes from TorT to TorS, leading to TorS activation. We identified several homologues of the TorT protein that define a new family of periplasmic binding proteins. We thus propose that the members of this family bind TMAO or related compounds and that they are involved in signal transduction or even substrate transport.

Hydrogen metabolism in the hyperthermophilic bacterium Aquifex aeolicus

Aquifex aeolicus is a microaerophilic, hydrogen-oxidizing, hyperthermophilic bacterium containing three [NiFe] hydrogenases. Two of these three enzymes (one membrane-bound and one soluble) have been purified and characterized. The Aquifex hydrogenases are thermostable and tolerant to oxygen. A cellular function for the three hydrogenases has been proposed. The two membrane-bound periplasmic hydrogenases may function in energy conservation, whereas the soluble cytoplasmic hydrogenase is probably involved in the CO(2) fixation pathway.

New Functional Sulfide Oxidase-Oxygen Reductase Supercomplex in the Membrane of the Hyperthermophilic Bacterium Aquifex aeolicus

Aquifex aeolicus, a hyperthermophilic and microaerophilic bacterium, obtains energy for growth from inorganic compounds alone. It was previously proposed that one of the respiratory pathways in this organism consists of the electron transfer from hydrogen sulfide (H2S) to molecular oxygen. H2S is oxidized by the sulfide quinone reductase, a membrane-bound flavoenzyme, which reduces the quinone pool. We have purified and characterized a novel membrane-bound multienzyme supercomplex that brings together all the molecular components involved in this bioenergetic chain. Our results indicate that this purified structure consists of one dimeric bc1 complex (complex III), one cytochrome c oxidase (complex IV), and one or two sulfide quinone reductases as well as traces of the monoheme cytochrome c555 and quinone molecules. In addition, this work strongly suggests that the cytochrome c oxidase in the supercomplex is a ba3-type enzyme. The supercomplex has a molecular mass of about 350 kDa and is enzymatically functional, reducing O2 in the presence of the electron donor, H2S. This is the first demonstration of the existence of such a respirasome carrying a sulfide oxidase-oxygen reductase activity. Moreover, the kinetic properties of the sulfide quinone reductase change slightly when integrated in the supercomplex, compared with the free enzyme. We previously purified a complete respirasome involved in hydrogen oxidation and sulfur reduction from Aquifex aeolicus. Thus, two different bioenergetic pathways (sulfur reduction and sulfur oxidation) are organized in this bacterium as supramolecular structures in the membrane. A model for the energetic sulfur metabolism of Aquifex aeolicus is proposed.

Mineral respiration under extreme acidic conditions: from a supramolecular organization to a molecular adaptation in Acidithiobacillus ferrooxidans

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic Gram-negative bacterium that can derive energy from the oxidation of ferrous iron at pH 2 using oxygen as electron acceptor. The study of this bacterium has economic and fundamental biological interest because of its use in the industrial extraction of copper and uranium from ores. For this reason, its respiratory chain has been analysed in detail in recent years. Studies have shown the presence of a functional supercomplex that spans the outer and the inner membranes and allows a direct electron transfer from the extracellular Fe2+ ions to the inner membrane cytochrome c oxidase. Iron induces the expression of two operons encoding proteins implicated in this complex as well as in the regeneration of the reducing power. Most of these are metalloproteins that have been characterized biochemically, structurally and biophysically. For some of them, the molecular basis of their adaptation to the periplasmic acidic environment has been described. Modifications in the metal surroundings have been highlighted for cytochrome c and rusticyanin, whereas, for the cytochrome c oxidase, an additional partner that maintains its stability and activity has been demonstrated recently.

The hyperthermophilic bacterium Aquifex aeolicus: from respiratory pathways to extremely resistant enzymes and biotechnological applications.

Aquifex aeolicus isolated from a shallow submarine hydrothermal system belongs to the order Aquificales which constitute an important component of the microbial communities at elevated temperatures. This hyperthermophilic chemolithoautotrophic bacterium, which utilizes molecular hydrogen, molecular oxygen, and inorganic sulfur compounds to flourish, uses the reductive TCA cycle for CO(2) fixation. In this review, the intricate energy metabolism of A. aeolicus is described. As the chemistry of sulfur is complex and multiple sulfur species can be generated, A. aeolicus possesses a multitude of different enzymes related to the energy sulfur metabolism. It contains also membrane-embedded [NiFe] hydrogenases as well as oxidases enzymes involved in hydrogen and oxygen utilization. We have focused on some of these proteins that have been extensively studied and characterized as super-resistant enzymes with outstanding properties. We discuss the potential use of hydrogenases in an attractive H(2)/O(2) biofuel cell in replacement of chemical catalysts. Using complete genomic sequence and biochemical data, we present here a global view of the energy-generating mechanisms of A. aeolicus including sulfur compounds reduction and oxidation pathways as well as hydrogen and oxygen utilization.

Reconstitution of supramolecular organization involved in energy metabolism at electrochemical interfaces for biosensing and bioenergy production

How the redox proteins and enzymes involved in bioenergetic pathways are organized is a relevant fundamental question, but our understanding of this is still incomplete. This review provides a critical examination of the electrochemical tools developed in recent years to obtain knowledge of the intramolecular and intermolecular electron transfer processes involved in metabolic pathways. Furthermore, better understanding of the electron transfer processes associated with energy metabolism will provide the basis for the rational design of biotechnological devices such as electrochemical biosensors, enzymatic and microbial fuel cells, and hydrogen production factories. Starting from the redox complexes involved in two relevant bacterial chains, i.e., from the hyperthermophile Aquifex aeolicus and the acidophile Acidithiobacillus ferrooxidans, examination of protein–protein interactions using electrochemistry is first reviewed, with a focus on the orientation of a protein on an electrochemical interface mimic of a physiological interaction between two partners. Special attention is paid to current research in the electrochemistry of essential membrane proteins, which is one mandatory step toward the understanding of energy metabolic pathways. The complex and challenging architectures built to reconstitute a membrane-like environment at an electrode are especially considered. The role played by electrochemistry in the attempt to consider full bacterial metabolism is finally emphasized through the study of whole cells immobilized at electrodes as suspensions or biofilms. Before the performances of biotechnological devices can be further improved to make them really attractive, questions remain to be addressed in this particular field of research. We discuss the bottlenecks that need to be overcome in the future.

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.