Antonio L. De Lacey

Laccase electrode for direct electrocatalytic reduction of O2 to H2O with high-operational stability and resistance to chloride inhibition

Laccase from Trametes hirsuta basidiomycete has been covalently bound to graphite electrodes electrochemically modified with phenyl derivatives as a way to attach the enzyme molecules with an adequate orientation for direct electron transfer (DET). Current densities up to 0.5mA/cm(2) of electrocatalytic reduction of O(2) to H(2)O were obtained in absence of redox mediators, suggesting preferential orientation of the T1 Cu centre of the laccase towards the electrode. The covalent attachment of the laccase molecules to the functionalized electrodes permitted remarkable operational stability. Moreover, O(2) bioelectroreduction based on DET between the laccase and the electrode was not inhibited by chloride ions, whereas mediated bioelectrocatalysis was. In contrast, fluoride ions inhibited both direct and mediated electron transfers-based bioelectrocatalytic reduction of O(2). Thus, two different modes of laccase inhibition by halides are discussed.

Gold Nanoparticles as Electronic Bridges for Laccase-Based Biocathodes

Direct electron transfer (DET) reactions between redox enzymes and electrodes can be maximized by oriented immobilization of the enzyme molecules onto an electroactive surface modified with functionalized gold nanoparticles (AuNPs). Here, we present such strategy for obtaining a DET-based laccase (Lc) cathode for O(2) electroreduction at low overpotentials. The stable nanostructured enzymatic electrode is based on the step-by-step covalent attachment of AuNPs and Lc molecules to porous graphite electrodes using the diazonium salt reduction strategy. Oriented immobilization of the enzyme molecules on adequately functionalized AuNPs allows establishing very fast DET with the electrode via their Cu T1 site. The measured electrocatalytic waves of O(2) reduction can be deconvoluted into two contributions. The one at lower overpotentials corresponds to immobilized Lc molecules that are efficiently wired by the AuNPs with a heterogeneous electron transfer rate constant k(0) ≫ 400 s(-1).

A membrane-, mediator-, cofactor-less glucose/oxygen biofuel cell

We report the fabrication and characterisation of a non-compartmentalised, mediator and cofactor free glucose-oxygen biofuel cell based on adsorbed enzymes exhibiting direct bioelectrocatalysis, viz. cellobiose dehydrogenase from Dichomera saubinetii and laccase from Trametes hirsuta as the anodic and cathodic bioelements, respectively, with the following characteristics: an open-circuit voltage of 0.73 V; a maximum power density of 5 microW cm(-2) at 0.5 V of the cell voltage and an estimated half-life of > 38 h in air-saturated 0.1 M citrate-phosphate buffer, pH 4.5 containing 5 mM glucose.

Introduction of Methionines in the Gas Channel Makes [NiFe] Hydrogenase Aero-Tolerant

Hydrogenases catalyze the conversion between 2H(+) + 2e(-) and H(2)(1). Most of these enzymes are inhibited by O(2), which represents a major drawback for their use in biotechnological applications. Improving hydrogenase O(2) tolerance is therefore a major contemporary challenge to allow the implementation of a sustainable hydrogen economy. We succeeded in improving O(2) tolerance, which we define here as the ability of the enzyme to resist for several minutes to O(2) exposure, by substituting with methionines small hydrophobic residues strongly conserved in the gas channel. Remarkably, the mutated enzymes remained active in the presence of an O(2) concentration close to that found in aerobic solutions in equilibrium with air, while the wild type enzyme is inhibited in a few seconds. Crystallographic and spectroscopic studies showed that the structure and the chemistry at the active site are not affected by the mutations. Kinetic studies demonstrated that the inactivation is slower and reactivation faster in these mutants. We propose that in addition to restricting O(2) diffusion to the active site of the enzyme, methionine may also interact with bound peroxide and provide an assisted escape route for H(2)O(2) toward the gas channel. These results show for the first time that it is possible to improve O(2)-tolerance of [NiFe] hydrogenases, making possible the development of biohydrogen production systems.

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.

Original design of an oxygen-tolerant [NiFe] hydrogenase: major effect of a valine-to-cysteine mutation near the active site.

Hydrogenases are efficient biological catalysts of H(2) oxidation and production. Most of them are inhibited by O(2), and a prerequisite for their use in biotechnological applications under air is to improve their oxygen tolerance. We have previously shown that exchanging the residue at position 74 in the large subunit of the oxygen-sensitive [NiFe] hydrogenase from Desulfovibrio fructosovorans could impact the reaction of the enzyme with O(2) (Dementin, S.; J. Am. Chem. Soc. 2009, 131, 10156-10164; Liebgott, P. P.; Nat. Chem. Biol. 2010, 6, 63-70). This residue, a valine in the wild-type enzyme, located at the bottleneck of the gas channel near the active site, has here been exchanged with a cysteine. A thorough characterization using a combination of kinetic, spectroscopic (EPR, FTIR), and electrochemical studies demonstrates that the V74C mutant has features of the naturally occurring oxygen-tolerant membrane-bound hydrogenases (MBH). The mutant is functional during several minutes under O(2), has impaired H(2)-production activity, and has a weaker affinity for CO than the WT. Upon exposure to O(2), it is converted into the more easily reactivatable inactive form, Ni-B, and this inactive state reactivates about 20 times faster than in the WT enzyme. Control experiments carried out with the V74S and V74N mutants indicate that protonation of the position 74 residue is not the reason the mutants reactivate faster than the WT enzyme. The electrochemical behavior of the V74C mutant toward O(2) is intermediate between that of the WT enzyme from D. fructosovorans and the oxygen-tolerant MBH from Aquifex aeolicus.

Electrochemical growth of Acidithiobacillus ferrooxidans on a graphite electrode for obtaining a biocathode for direct electrocatalytic reduction of oxygen

An aspect in microbial fuel cell research that is currently of great interest is the development of bacterial cathodes. Bacterial cathodes that catalyze oxygen reduction to water at low pH have the advantage of overcoming the kinetic limitations due to the requirement of 4 protons per molecule reduced. In this work we have studied the performance of a biocathode using as electrocatalyst an acidophile microorganism: Acidithiobacillus ferrooxidans. Growth of the microorganism directly on the electrode took place using an applied voltage of 0 V vs. SCE as the only energy source and without adding redox mediators to the solution. Current densities of up to 5 A m(-2) were measured for O2 reduction in the At. ferrooxidans cathode at pH 2.0 and the electrocatalytic wave was shifted 300 mV to higher potential compared to the control graphite electrodes without the bacterium.

Understanding and Tuning the Catalytic Bias of Hydrogenase

When enzymes are optimized for biotechnological purposes, the goal often is to increase stability or catalytic efficiency. However, many enzymes reversibly convert their substrate and product, and if one is interested in catalysis in only one direction, it may be necessary to prevent the reverse reaction. In other cases, reversibility may be advantageous because only an enzyme that can operate in both directions can turnover at a high rate even under conditions of low thermodynamic driving force. Therefore, understanding the basic mechanisms of reversibility in complex enzymes should help the rational engineering of these proteins. Here, we focus on NiFe hydrogenase, an enzyme that catalyzes H(2) oxidation and production, and we elucidate the mechanism that governs the catalytic bias (the ratio of maximal rates in the two directions). Unexpectedly, we found that this bias is not mainly determined by redox properties of the active site, but rather by steps which occur on sites of the proteins that are remote from the active site. We evidence a novel strategy for tuning the catalytic bias of an oxidoreductase, which consists in modulating the rate of a step that is limiting only in one direction of the reaction, without modifying the properties of the active site.

Preferential Use of an Anode as an Electron Acceptor by an Acidophilic Bacterium in the Presence of Oxygen

ABSTRACT Several anaerobic metal-reducing bacteria have been shown to be able to donate electrons directly to an electrode. This property is of great interest for microbial fuel cell development. To date, microbial fuel cell design requires avoiding O2 diffusion from the cathodic compartment to the sensitive anodic compartment. Here, we show that Acidiphilium sp. strain 3.2 Sup 5 cells that were isolated from an extreme acidic environment are able to colonize graphite felt electrodes. These bacterial electrodes were able to produce high-density electrocatalytic currents, up to 3 A/m2 at a poised potential of +0.15 V (compared to the value for the reference standard calomel electrode) in the absence of redox mediators, by oxidizing glucose even at saturating air concentrations and very low pHs.

O2-independent formation of the inactive states of NiFe hydrogenase

We studied the mechanism of aerobic inactivation of Desulfovibrio fructosovorans nickel-iron (NiFe) hydrogenase by quantitatively examining the results of electrochemistry, EPR and FTIR experiments. They suggest that, contrary to the commonly accepted mechanism, the attacking O(2) is not incorporated as an active site ligand but, rather, acts as an electron acceptor. Our findings offer new ways toward the understanding of O(2) inactivation and O(2) tolerance in NiFe hydrogenases.