Carbon Fixation in the Chemolithoautotrophic Bacterium Aquifex aeolicus Involves Two Low-Potential Ferredoxins as Partners of the PFOR and OGOR Enzymes.

Aquifex aeolicus is a microaerophilic hydrogen- and sulfur -oxidizing bacterium that assimilates CO2 via the reverse tricarboxylic acid cycle (rTCA). Key enzymes of this pathway are pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OGOR), which are responsible, respectively, for the reductive carboxylation of acetyl-CoA to pyruvate and of succinyl-CoA to 2-oxoglutarate, two energetically unfavorable reactions that require a strong reduction potential. We have confirmed, by biochemistry and proteomics, that A. aeolicus possesses a pentameric version of these enzyme complexes ((αßγδε)2) and that they are highly abundant in the cell. In addition, we have purified and characterized, from the soluble fraction of A. aeolicus, two low redox potential and oxygen-stable [4Fe-4S] ferredoxins (Fd6 and Fd7, E0 = -440 and -460 mV, respectively) and shown that they can physically interact and exchange electrons with both PFOR and OGOR, suggesting that they could be the physiological electron donors of the system in vivo. Shotgun proteomics indicated that all the enzymes assumed to be involved in the rTCA cycle are produced in the A. aeolicus cells. A number of additional enzymes, previously suggested to be part of a putative partial Wood-Ljungdahl pathway used for the synthesis of serine and glycine from CO2 were identified by mass spectrometry, but their abundance in the cell seems to be much lower than that of the rTCA cycle. Their possible involvement in carbon assimilation is discussed.

Hydrogenase-based electrode for hydrogen sensing in a fermentation bioreactor.

The hydrogen-based economy will require not only sustainable hydrogen production but also sensitive and cheap hydrogen sensors. Commercially available H2 sensors are limited by either use of noble metals or elevated temperatures. In nature, hydrogenase enzymes present high affinity and selectivity for hydrogen, while being able to operate in mild conditions. This study aims at evaluating the performance of an electrochemical sensor based on carbon nanomaterials with immobilised hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus for H2 detection. The effect of various parameters, including the surface chemistry, dispersion degree and amount of deposited carbon nanotubes, enzyme concentration, temperature and pH on the H2 oxidation are investigated. Although the highest catalytic response is obtained at a temperature around 60 °C, a noticeable current can be obtained at room temperature with a low amount of protein less than 1 µM. An original pulse-strategy to ensure H2 diffusion to the bioelectrode allows to reach H2 sensitivity of 4 µA cm-2 per % H2 and a linear range between 1 and 20%. Sustainable hydrogen was then produced through dark fermentation performed by a synthetic bacterial consortium in an up-flow anaerobic packed-bed bioreactor. Thanks to the outstanding properties of the A. aeolicus hydrogenase, the biosensor was demonstrated to be quite insensitive to CO2 and H2S produced as the main co-products of the bioreactor. Finally, the bioelectrode was used for the in situ measurement of H2 produced in the bioreactor in steady-state.

Local pH Modulation during Electro-Enzymatic O2 Reduction: Characterization of the Influence of Ionic Strength by In Situ Fluorescence Microscopy.

Understanding how environmental factors affect the bioelectrode efficiency and stability is of uttermost importance to develop high-performance bioelectrochemical devices. By coupling fluorescence confocal microscopy in situ to electrochemistry, this work focuses on the influence of the ionic strength on electro-enzymatic catalysis. In this context, the 4 e-/4 H+ reduction of O2 into water by the bilirubin oxidase from Myrothecium verrucaria (MvBOD) is considered as a model. The effects of salt concentration on the enzyme activity and stability were probed by enzymatic assays performed in homogeneous catalysis conditions and monitored by UV-vis absorption spectroscopy. They were also investigated in heterogeneous catalysis conditions by electrochemical measurements with MvBOD immobilized at a graphite microelectrode. We demonstrate that the catalytic activity and stability of the enzyme both in solution and in the immobilized state at the bioelectrode were conserved with an electrolyte concentration of up to 0.5 M, both in a buffered and a non-buffered electrolyte. Relying on this, we used fluorescence confocal laser scanning microscopy coupled in situ to electrochemistry to explore the local pH of the electrolyte at the vicinity of the electrode surface at various ionic strengths and for several overpotentials. 3D proton depletion profiles generated by the interfacial electro-enzymatic reaction were recorded in the presence of a pH-sensitive fluorophore. These concentration profiles were shown to contract with increasing ionic strength, thus highlighting the need for a minimal electrolyte concentration to ensure availability of charged substrates at the electrode surface during electro-enzymatic experiments.

A periplasmic cupredoxin with a green CuT1.5 center is involved in bacterial copper tolerance.

The importance of copper resistance pathways in pathogenic bacteria is now well recognized, since macrophages use copper to fight bacterial infections. Additionally, considering the increase of antibiotic resistance, growing attention is given to the antimicrobial properties of copper. It is of primary importance to understand how bacteria deal with copper. The Cu-resistant cuproprotein CopI is present in many human bacterial pathogens and environmental bacteria and crucial under microaerobiosis (conditions for most pathogens to thrive within their host). Hence, understanding its mechanism of function is essential. CopI proteins share conserved histidine, cysteine, and methionine residues that could be ligands for different copper binding sites, among which the cupredoxin center could be involved in the protein function. Here, we demonstrated that Vibrio cholerae and Pseudomonas aeruginosa CopI restore the Cu-resistant phenotype in the Rubrivivax gelatinosus ΔcopI mutant. We identified that Cys125 (ligand in the cupredoxin center) and conserved histidines and methionines are essential for R. gelatinosus CopI (RgCopI) function. We also performed spectroscopic analyses of the purified RgCopI protein and showed that it is a green cupredoxin able to bind a maximum of three Cu(II) ions: (i) a green Cu site (CuT1.5), (ii) a type 2 Cu binding site (T2) located in the N-terminal region, and (iii) a third site with a yet unidentified location. CopI is therefore one member of the poorly described CuT1.5 center cupredoxin family. It is unique, since it is a single-domain cupredoxin with more than one Cu site involved in Cu resistance.

Mutations in the coordination spheres of T1 Cu affect Cu2+-activation of the laccase from Thermus thermophilus.

Thermus thermophilus laccase belongs to the sub-class of multicopper oxidases that is activated by the extra binding of copper to a methionine-rich domain allowing an electron pathway from the substrate to the conventional first electron acceptor, the T1 Cu. In this work, two key amino acid residues in the 1st and 2nd coordination spheres of T1 Cu are mutated in view of tuning their redox potential and investigating their influence on copper-related activity. Evolution of the kinetic parameters after copper addition highlights that both mutations play a key role influencing the enzymatic activity in distinct unexpected ways. These results clearly indicate that the methionine rich domain is not the only actor in the cuprous oxidase activity of CueO-like enzymes.

The pH-Induced Selectivity Between Cysteine or Histidine Coordinated Heme in an Artificial α-Helical Metalloprotein.

De Novo metalloprotein design assesses the relationship between metal active site architecture and catalytic reactivity. Herein, we use an α-helical scaffold to control the iron coordination geometry when a heme cofactor is allowed to bind to either histidine or cysteine ligands, within a single artificial protein. Consequently, we uncovered a reversible pH-induced switch of the heme axial ligation within this simplified scaffold. Characterization of the specific heme coordination modes was done by using UV/Vis and Electron Paramagnetic Resonance spectroscopies. The penta- or hexa-coordinate thiolate heme (9≤pH≤11) and the penta-coordinate imidazole heme (6≤pH≤8.5) reproduces well the heme ligation in chloroperoxidases or cyt P450 monooxygenases and peroxidases, respectively. The stability of heme coordination upon ferric/ferrous redox cycling is a crucial property of the construct. At basic pHs, the thiolate mini-heme protein can catalyze O2 reduction when adsorbed onto a pyrolytic graphite electrode.

Implicit Modeling of the Impact of Adsorption on Solid Surfaces for Protein Mechanics and Activity with a Coarse-Grained Representation.

Surface immobilized enzymes play a key role in numerous biotechnological applications such as biosensors, biofuel cells, or biocatalytic synthesis. As a consequence, the impact of adsorption on the enzyme structure, dynamics, and function needs to be understood on the molecular level as it is critical for the improvement of these technologies. With this perspective in mind, we used a theoretical approach for investigating local protein flexibility on the residue scale that couples a simplified protein representation with an elastic network and Brownian dynamics simulations. The impact of protein adsorption on a solid surface is implicitly modeled via additional external constraints between the residues in contact with the surface. We first performed calculations on a redox enzyme, bilirubin oxidase (BOD) from M. verrucaria, to study the impact of adsorption on its mechanical properties. The resulting rigidity profiles show that, in agreement with the available experimental data, the mechanical variations observed in the adsorbed BOD will depend on its orientation and its anchor residues (i.e., residues that are in contact with the functionalized surface). Additional calculations on ribonuclease A and nitroreductase shed light on how seemingly stable adsorbed enzymes can nonetheless display an important decrease in their catalytic activity resulting from a perturbation of their mechanics and internal dynamics.

Nanosecond Laser-Fabricated Monolayer of Gold Nanoparticles on ITO for Bioelectrocatalysis.

Redox enzymes can be envisioned as biocatalysts in various electrocatalytic-based devices. Among factors that play roles in bioelectrochemistry limitations, the effect of enzyme-enzyme neighboring interaction on electrocatalysis has rarely been investigated, although critical in vivo. We report in this work an in-depth study of gold nanoparticles prepared by laser ablation in the ultimate goal of determining the relationship between activity and enzyme density on electrodes. Nanosecond laser interaction with nanometric gold films deposited on indium tin oxide support was used to generate in situ gold nanoparticles (AuNPs) free from any stabilizers. A comprehensive analysis of AuNP size and coverage, as well as total geometric surface vs. electroactive surface is provided as a function of the thickness of the treated gold layer. Using microscopy and electrochemistry, the long-term stability of AuNP-based electrodes in the atmosphere and in the electrolyte is demonstrated. AuNPs formed by laser treatment are then modified by thiol chemistry and their electrochemical behavior is tested with a redox probe. Finally, enzyme adsorption and bioelectrocatalysis are evaluated in the case of two enzymes, i.e., the Myrothecium verrucaria bilirubin oxidase and the Thermus thermophilus laccase. Behaving differently on charged surfaces, they allow demonstrating the validity of laser treated AuNPs for bioelectrocatalysis.

In Situ Fluorescence Tomography Enables a 3D Mapping of Enzymatic O2 Reduction at the Electrochemical Interface.

Getting information about the fate of immobilized enzymes and the evolution of their environment during turnover is a mandatory step toward bioelectrode optimization for effective use in biodevices. We demonstrate here the proof-of-principle visual characterization of the reactivity at an enzymatic electrode thanks to fluorescence confocal laser scanning microscopy (FCLSM) implemented in situ during the electrochemical experiment. The enzymatic O2 reduction involves proton-coupled electron transfers. Therefore, fluorescence variation of a pH-dependent fluorescent dye in the electrode vicinity enables reaction visualization. Simultaneous collection of electrochemical and fluorescence signals gives valuable space- and time-resolved information. Once the technical challenges of such a coupling are overcome, in situ FCLSM affords a unique way to explore reactivity at the electrode surface and in the electrolyte volume. Unexpected features are observed, especially the pH evolution of the enzyme environment, which is also indicated by a characteristic concentration profile within the diffusion layer. This coupled approach also gives access to a cartography of the electrode surface response (i.e., heterogeneity), which cannot be obtained solely by an electrochemical means.

Controllable Display of Sequential Enzymes on Yeast Surface with Enhanced Biocatalytic Activity toward Efficient Enzymatic Biofuel Cells.

A precisely localized enzyme cascade was constructed by integrating two sequential enzymes, glucoamylase (GA) and glucose oxidase (GOx), on a yeast cell surface through an a-agglutinin receptor as the anchoring motif with cohesin-dockerin interaction. The overall catalytic activities of the combinant strains were significantly dependent on the assembly method, enzyme molecular size, enzyme order, and enzyme stoichiometry. The combinant strain with GA-DocC initially bound scaffoldin prior to GOx-DocT exhibited a higher overall reaction rate. The highest overall reaction rate (29.28 ± 1.15 nmol H2O2 min-1mL-1) was achieved when GA/GOx ratio was 2:1 with enzyme order: yeast-GA-GOx-GA, 4-fold enhancement compared to free enzyme mixture. Further, the first example of starch/O2 enzymatic biofuel cells (EBFCs) using codisplayed GA/GOx based bioanodes were assembled, demonstrating excellent direct biomass-to-electricity conversion. The optimized EBFC registered an open-circuit voltage of 0.78 V and maximum power density (Pmax) of 36.1 ± 2.5 µW cm-2, significantly higher than the Pmax for other starch/O2 EBFCs reported so far. Therefore, this work highlights rational organization of sequential enzymes for enhanced biocatalytic activity and stability, which would find applications in biocatalysis, enzymatic biofuel cells, biosensing, and bioelectro-synthesis.

Interplay between Orientation at Electrodes and Copper Activation of Thermus thermophilus Laccase for O2 Reduction.

Multicopper oxidases (MCOs) catalyze the oxidation of a variety of substrates while reducing oxygen into water through four copper atoms. As an additional feature, some MCOs display an enhanced activity in solution in the presence of Cu2+. This is the case of the hyperthermophilic laccase HB27 from Thermus thermophilus, the physiologic role of which is unknown. As a particular feature, this enzyme presents a methionine rich domain proposed to be involved in copper interaction. In this work, laccase from T. thermophilus was produced in E. coli, and the effect of Cu2+ on its electroactivity at carbon nanotube modified electrodes was investigated. Direct O2 electroreduction is strongly dictated by carbon nanotube surface chemistry in accordance with the enzyme dipole moment. In the presence of Cu2+, an additional low potential cathodic wave occurs, which was never described earlier. Analysis of this wave as a function of Cu2+ availability allows us to attribute this wave to a cuprous oxidase activity displayed by the laccase and induced by copper binding close to the Cu T1 center. A mutant lacking the methionine-rich hairpin domain characteristic of this laccase conserves its copper activity suggesting a different site of copper binding. This study provides new insight into the copper effect in methionine rich MCOs and highlights the utility of the electrochemical method to investigate cuprous oxidase activity and to understand the physiological role of these MCOs.

Bacterial Respiratory Chain Diversity Reveals a Cytochrome c Oxidase Reducing O2 at Low Overpotentials.

Cytochrome c oxidases (CcOs) are the terminal enzymes in energy-converting chains of microorganisms, where they reduce oxygen into water. Their affinity for O2 makes them attractive biocatalysts for technological devices in which O2 concentration is limited, but the high overpotentials they display on electrodes severely limit their applicative use. Here, the CcO of the acidophilic bacterium Acidithiobacillus ferrooxidans is studied on various carbon materials by direct protein electrochemistry and mediated one with redox mediators either diffusing or co-immobilized at the electrode surface. The entrapment of the CcO in a network of hydrophobic carbon nanofibers permits a direct electrochemical communication between the enzyme and the electrode. We demonstrate that the CcO displays a µM affinity for O2 and reduces O2 at exceptionally high electrode potentials in the range of +700 to +540 mV vs NHE over a pH range of 4-6. The kinetics of interactions between the enzyme and its physiological partners are fully quantified. Based on these results, an electron transfer pathway allowing O2 reduction in the acidic metabolic chain is proposed.

Tackling the Challenges of Enzymatic (Bio)Fuel Cells.

The ever-increasing demands for clean and sustainable energy sources combined with rapid advances in biointegrated portable or implantable electronic devices have stimulated intensive research activities in enzymatic (bio)fuel cells (EFCs). The use of renewable biocatalysts, the utilization of abundant green, safe, and high energy density fuels, together with the capability of working at modest and biocompatible conditions make EFCs promising as next generation alternative power sources. However, the main challenges (low energy density, relatively low power density, poor operational stability, and limited voltage output) hinder future applications of EFCs. This review aims at exploring the underlying mechanism of EFCs and providing possible practical strategies, methodologies and insights to tackle these issues. First, this review summarizes approaches in achieving high energy densities in EFCs, particularly, employing enzyme cascades for the deep/complete oxidation of fuels. Second, strategies for increasing power densities in EFCs, including increasing enzyme activities, facilitating electron transfers, employing nanomaterials, and designing more efficient enzyme-electrode interfaces, are described. The potential of EFCs/(super)capacitor combination is discussed. Third, the review evaluates a range of strategies for improving the stability of EFCs, including the use of different enzyme immobilization approaches, tuning enzyme properties, designing protective matrixes, and using microbial surface displaying enzymes. Fourth, approaches for the improvement of the cell voltage of EFCs are highlighted. Finally, future developments and a prospective on EFCs are envisioned.

Influence of cytochrome charge and potential on the cathodic current of electroactive artificial biofilms.

An electroactive artificial biofilm has been optimized for the cathodic reduction of fumarate by Shewanella oneidensis. The system is based on the self-assembly of multi-walled carbon nanotubes with bacterial cells in the presence of a c-type cytochrome. The aggregates are then deposited on an electrode to form the electroactive artificial biofilm. Six c-type cytochromes have been studied, from bovine heart or Desulfuromonas and Desulfuvibrio strains. The isoelectric point of the cytochrome controls the self-assembly process that occurs only with positively-charged cytochromes. The redox potential of the cytochrome is critical for electron transfer reactions with membrane cytochromes of the Mtr pathway. Optimal results have been obtained with c3 from Desulfovibrio vulgaris Hildenborough having an isoelectric point of 10.2 and redox potentials of the four hemes ranging between -290 and -375 mV vs SHE. A current density of 170 µA cm-2 could be achieved in the presence of 50 mM fumarate. The stability of the electrochemical response was evaluated, showing a regular decrease of the current within 13 h, possibly due to the inactivation or leaching of loosely-bound cytochromes from the biofilm.

Interprotein Electron Transfer between FeS-Protein Nanowires and Oxygen-Tolerant NiFe Hydrogenase.

Self-assembled redox protein nanowires have been exploited as efficient electron shuttles for an oxygen-tolerant hydrogenase. An intra/inter-protein electron transfer chain has been achieved between the iron-sulfur centers of rubredoxin and the FeS cluster of [NiFe] hydrogenases. [NiFe] Hydrogenases entrapped in the intricated matrix of metalloprotein nanowires achieve a stable, mediated bioelectrocatalytic oxidation of H2 at low-overpotential.

Impact of copper ligand mutations on a cupredoxin with a green copper center.

Mononuclear cupredoxins contain a type 1 copper center with a trigonal or tetragonal geometry usually maintained by four ligands, a cystein, two histidines and a methionine. The recent discovery of new members of this family with unusual properties demonstrates, however, the versatility of this class of proteins. Changes in their ligand set lead to drastic variation in their metal site geometry and in the resulting spectroscopic and redox features. In our work, we report the identification of the copper ligands in the recently discovered cupredoxin AcoP. We show that even though AcoP possesses a classical copper ligand set, it has a highly perturbed copper center. In depth studies of mutant's properties suggest a high degree of constraint existing in the copper center of the wild type protein and even the addition of exogenous ligands does not lead to the reconstitution of the initial copper center. Not only the chemical nature of the axial ligand but also constraints brought by its covalent binding to the protein backbone might be critical to maintain a green copper site with high redox potential. This work illustrates the importance of experimentally dissecting the molecular diversity of cupredoxins to determine the molecular determinants responsible for their copper center geometry and redox potential.

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 ß.

How the Intricate Interactions between Carbon Nanotubes and Two Bilirubin Oxidases Control Direct and Mediated O2 Reduction.

Due to the lack of a valid approach in the design of electrochemical interfaces modified with enzymes for efficient catalysis, many oxidoreductases are still not addressed by electrochemistry. We report in this work an in-depth study of the interactions between two different bilirubin oxidases, (from the fungus Myrothecium verrucaria and from the bacterium Bacillus pumilus), catalysts of oxygen reduction, and carbon nanotubes bearing various surface charges (pristine, carboxylic-, and pyrene-methylamine-functionalized). The surface charges and dipole moment of the enzymes as well as the surface state of the nanomaterials are characterized as a function of pH. An original electrochemical approach allows determination of the best interface for direct or mediated electron transfer processes as a function of enzyme, nanomaterial type, and adsorption conditions. We correlate these experimental results to theoric voltammetric curves. Such an integrative study suggests strategies for designing efficient bioelectrochemical interfaces toward the elaboration of biodevices such as enzymatic fuel cells for sustainable electricity production.