Tuning Chemoenzymatic Pd/Laccase Conformation Toward Optimized Heterogeneous Aerobic Oxidation.

Heterogeneous chemoenzymatic catalysts differing in their spatial organization and relative orientation of their enzymatic laccase and Pd units confined into macrocellular silica foams were tested on veratryl alcohol oxidation. When operating under continuous flow, we show that the catalytic efficiency of hybrids is significantly enhanced when the Pd(II) complex is combined with a laccase exhibiting a surface located lysine next to the T1 oxidation site of the enzyme.

Methionine oxidation of carbohydrate-active enzymes during white-rot wood decay.

White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well documented, the impact of ROS on the oxidation of the secreted proteins remains unclear, and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H218O2 with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed the identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.IMPORTANCEThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H2O2) on secreted proteins remains unclear. This study aims at evaluating the effect of H2O2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot fungi Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H2O2. The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met, like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown.

Investigation of dirigent like domains from bacterial genomes.

BACKGROUND: DIRs are mysterious protein that have the ability to scavenge free radicals, which, are highly reactive with molecules in their vicinity. What is even more fascinating is that they carry out from these highly unstable species, a selective reaction (i.e., stereoenantioselective) from a well-defined substrate to give a very precise product. Unfortunately, to date, only three products have been demonstrated following studies on DIRs from the plant world, which until now was the kingdom where these proteins had been demonstrated. Within this kingdom, each DIR protein has its own type of substrate. The products identified to date, have on the other hand, a strong economic impact: in agriculture for example, the biosynthesis of (+)-gossypol could be highlighted (a repellent antifood produced by the cotton plant) by the DIRs of cotton. In forsythia plant species, it is the biosynthesis of (-)-pinoresinol, an intermediate leading to the synthesis of podophyllotoxine (a powerful anicancerous agent) which has been revealed. Recently, a clear path of study, potentially with strong impact, appeared by the hypothesis of the potential existence of protein DIR within the genomes of prokaryotes. The possibility of working with this type of organism is an undeniable advantage: since many sequenced genomes are available and the molecular tools are already developed. Even easier to implement and working on microbes, of less complex composition, offers many opportunities for laboratory studies. On the other hand, the diversity of their environment (e.g., soil, aquatic environments, extreme environmental conditions (pH, temperature, pressure) make them very diverse and varied subjects of study. Identifying new DIR proteins from bacteria means identifying new substrate or product molecules from these organisms. It is the promise of going further in understanding the mechanism of action of these proteins and this will most likely have a strong impact in the fields of agricultural, pharmaceutical and/or food chemistry. RESULTS: Our goal is to obtain as much information as possible about these proteins to unlock the secrets of their exceptional functioning. Analyzes of structural and functional genomic data led to the identification of the Pfam PF03018 domain as characteristic of DIR proteins. This domain has been further identified in the sequence of bacterial proteins therefore named as DIR-like (DIRL). We have chosen a multidisciplinary bioinformatic approach centered on bacterial genome identification, gene expression and regulation signals, protein structures, and their molecular information content. The objective of this study was to perform a thorough bioinformatic analysis on these DIRLs to highlight any information leading to the selection of candidate bacteria for further cloning, purification, and characterization of bacterial DIRs. CONCLUSIONS: From studies of DIRL genes identification, primary structures, predictions of their secondary and tertiary structures, prediction of DIRL signals sequences, analysis of their gene organization and potential regulation, a list of primary bacterial candidates is proposed.

2D and 3D maximum-quantum NMR and diffusion spectroscopy for the characterization of enzymatic reaction mixtures.

1D 1H NMR spectroscopy has been widely used to monitor enzymatic activity by recording the evolution of the spectra of substrates and/or products, thanks to the linear response of NMR. For complex systems involving the coexistence of multiple compounds (substrate, final product and various intermediates), the identification and quantification can be a more arduous task. Here, we present a simple analytical method for the rapid characterization of reaction mixtures involving enzymatic complexes using Maximum Quantum (MaxQ) NMR, accelerated with the Non-Uniform Sampling (NUS) acquisition procedure. Specifically, this approach enables, in the first analytical step, the counting of the molecules present in the samples. We also show, using two different enzymatic systems, that the implementation of these pulse sequences implies precautions related to the short relaxation times due to the presence of metallo-enzymes or paramagnetic catalysts. Finally, the combination of MaxQ and diffusion experiments, which leads to a 3D chart, greatly improves the resolution and offers an extreme simplification of the spectra while giving valuable indications on the affinity of the enzymes to the different compounds present in the reaction mixture.

Coniferyl Alcohol Radical Detection by the Dirigent Protein AtDIR6 Monitored by EPR.

Plant dirigent proteins (DIRs) control the stereoselectivity of the monolignol coniferyl alcohol radical coupling. The main mechanistic hypothesis on this chemo- and stereoselective reaction invokes a binding of coniferyl alcohol radical substrates in the dirigent protein active site so that only one enantiomeric form can be produced. We have studied the influence of the Arabidopsis thaliana AtDIR6 protein on the transient coniferyl alcohol radical by EPR. Herein, we show that AtDIR6 stabilizes coniferyl alcohol radicals prior to directing their coupling towards the formation of (-)-pinoresinol.

Clicked Bifunctional Dendrimeric and Cyclopeptidic Addressable Redox Scaffolds for the Functionalization of Carbon Nanotubes with Redox Molecules and Enzymes.

Carbon nanotube electrodes were modified with ferrocene and laccase using two different click reactions strategies and taking advantage of bifunctional dendrimers and cyclopeptides. Using diazonium functionalization and the efficiency of oxime ligation, the combination of both multiwalled carbon nanotube surfaces and modified dendrimers or cyclopeptides allows the access to a high surface coverage of ferrocene in the order of 50 nmol cm-2, a 50-fold increase compared to a classic click reaction without oxime ligation of these highly branched macromolecules. Furthermore, this original immobilization strategy allows the immobilization of mono- and bi-functionalized active multicopper enzymes, laccases, via copper(I)-catalyzed azide-alkyne cycloaddition. Electrochemical studies underline the high efficiency of the oxime-ligated dendrimers or cyclopeptides for the immobilization of redox entities on surfaces while being detrimental to electron tunneling with enzyme active sites despite controlled orientation.

Efficiency of Site-Specific Clicked Laccase-Carbon Nanotubes Biocathodes towards O2 Reduction.

A maximization of a direct electron transfer (DET) between redox enzymes and electrodes can be obtained through the oriented immobilization of enzymes onto an electroactive surface. Here, a strategy for obtaining carbon nanotube (CNTs) based electrodes covalently modified with perfectly control-oriented fungal laccases is presented. Modelizations of the laccase-CNT interaction and of electron conduction pathways serve as a guide in choosing grafting positions. Homogeneous populations of alkyne-modified laccases are obtained through the reductive amination of a unique surface-accessible lysine residue selectively engineered near either one or the other of the two copper centers in enzyme variants. Immobilization of the site-specific alkynated enzymes is achieved by copper-catalyzed click reaction on azido-modified CNTs. A highly efficient reduction of O2 at low overpotential and catalytic current densities over -3 mA cm-2 are obtained by minimizing the distance from the electrode surface to the trinuclear cluster.

A Reversible Electron Relay to Exclude Sacrificial Electron Donors in the Photocatalytic Oxygen Atom Transfer Reaction with O2 in Water.

Using light energy and O2 for the direct chemical oxidation of organic substrates is a major challenge. A limitation is the use of sacrificial electron donors to activate O2 by reductive quenching of the photosensitizer, generating undesirable side products. A reversible electron acceptor, methyl viologen, can act as electron shuttle to oxidatively quench the photosensitizer, [Ru(bpy)3 ]2+ , generating the highly oxidized chromophore and the powerful reductant methyl-viologen radical MV+. . MV+. can then reduce an iron(III) catalyst to the iron(II) form and concomitantly O2 to O2.- in an aqueous medium to generate an active iron(III)-(hydro)peroxo species. The oxidized photosensitizer is reset to its ground state by oxidizing an alkene substrate to an alkenyl radical cation. Closing the loop, the reaction of the iron reactive intermediate with the substrate or its radical cation leads to the formation of two oxygenated compounds, the diol and the aldehyde following two different pathways.

Electrochemical Water Oxidation and Stereoselective Oxygen Atom Transfer Mediated by a Copper Complex.

Water oxidation by copper-based complexes to form dioxygen has attracted attention in recent years, with the aim of developing efficient and cheap catalysts for chemical energy storage. In addition, high-valent metal-oxo species produced by the oxidation of metal complexes in the presence of water can be used to achieve substrate oxygenation with the use of H2 O as an oxygen source. To date, this strategy has not been reported for copper complexes. Herein, a copper(II) complex, [(RPY2)Cu(OTf)2 ] (RPY2=N-substituted bis[2-pyridyl(ethylamine)] ligands; R=indane; OTf=triflate), is used. This complex, which contains an oxidizable substrate moiety (indane), is used as a tool to monitor an intramolecular oxygen atom transfer reaction. Electrochemical properties were investigated and, upon electrolysis at 1.30 V versus a normal hydrogen electrode (NHE), both dioxygen production and oxygenation of the indane moiety were observed. The ligand was oxidized in a highly diastereoselective manner, which indicated that the observed reactivity was mediated by metal-centered reactive species. The pH dependence of the reactivity was monitored and correlated with speciation deduced from different techniques, ranging from potentiometric titrations to spectroscopic studies and DFT calculations. Water oxidation for dioxygen production occurs at neutral pH and is probably mediated by the oxidation of a mononuclear copper(II) precursor. It is achieved with a rather low overpotential (280 mV at pH 7), although with limited efficiency. On the other hand, oxygenation is maximum at pH 8-8.5 and is probably mediated by the electrochemical oxidation of an antiferromagnetically coupled dinuclear bis(µ-hydroxo) copper(II) precursor. This constitutes the first example of copper-centered oxidative water activation for a selective oxygenation reaction.

Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis.

The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.

Characterization of Cu(II)-reconstituted ACC Oxidase using experimental and theoretical approaches.

1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a non heme iron(II) containing enzyme that catalyzes the final step of the ethylene biosynthesis in plants. The iron(II) ion is bound in a facial triad composed of two histidines and one aspartate (H177, D179 and H234). Several active site variants were generated to provide alternate binding motifs and the enzymes were reconstituted with copper(II). Continuous wave (cw) and pulsed Electron Paramagnetic Resonance (EPR) spectroscopies as well as Density Functional Theory (DFT) calculations were performed and models for the copper(II) binding sites were deduced. In all investigated enzymes, the copper ion is equatorially coordinated by the two histidine residues (H177 and H234) and probably two water molecules. The copper-containing enzymes are inactive, even when hydrogen peroxide is used in peroxide shunt approach. EPR experiments and DFT calculations were undertaken to investigate substrate's (ACC) binding on the copper ion and the results were used to rationalize the lack of copper-mediated activity.

Visible light-driven O2 reduction by a porphyrin-laccase system.

Several recent studies have shown that the combination of photosensitizers with metalloenzymes can support a light-driven multielectron reduction of molecules such as CO(2) or HCN. Here we show that the association of the zinc tetramethylpyridinium porphyrin (ZnTMPyP(4+)) photosensitizer with the multicopper oxidase (MCO) laccase allows to link the oxidation of an organic molecule to the four electrons reduction of dioxygen into water. The enzyme is photoreduced within minutes with porphyrin/enzyme ratio as low as 1:40. With a 1:1 ratio, the dioxygen consumption rate is 1.7 µmol L(-1) s(-1). Flash photolysis experiments support the formation of the triplet excited state of ZnTMPyP(4+) which reduces the enzyme to form a radical cation of the porphyrin with a k(ET) ≈ 10(7) s(-1) M(-1). The long-lived triplet excited state of the ZnTMPyP(4+) (τ(0) = 0.72 ms) accounts for a substantial electron-transfer quantum yield, φ(ET) = 0.35. Consequently, the enzyme-dependent photo-oxidation of the electron donor occurs with a turnover of 8 min(-1) for the one-electron oxidation process, thereby supporting the suitability of such enzyme/sensitizer hybrid systems for aerobic photodriven transformations on substrates. This study is the first example of a phorphyrin-sensitized four-electron reduction of an enzyme of the MCO family, leading to photoreduction of dioxygen into water.

Structural and magnetic characterization of a tetranuclear copper(II) cubane stabilized by intramolecular metal cation-π interactions.

A novel tetranuclear copper(II) complex (1) was synthesized from the self-assembly of copper(II) perchlorate and the ligand N-benzyl-1-(2-pyridyl)methaneimine (L(1)). Single-crystal X-ray diffraction studies revealed that complex 1 consists of a Cu4(OH)4 cubane core, where the four copper(II) centers are linked by µ3-hydroxo bridges. Each copper(II) ion is in a distorted square-pyramidal geometry. X-ray analysis also evidenced an unusual metal cation-π interaction between the copper ions and phenyl substituents of the ligand. Calculations based on the density functional theory method were used to quantify the strength of this metal-π interaction, which appears as an important stabilizing parameter of the cubane core, possibly acting as a driving parameter in the self-aggregation process. In contrast, using the ligand N-phenethyl-1-(2-pyridyl)methaneimine (L(2)), which only differs from L(1) by one methylene group, the same synthetic procedure led to a binuclear bis(µ-hydroxo)copper(II) complex (2) displaying intermolecular π-π interactions or, by a slight variation of the experimental conditions, to a mononuclear complex (3). These complexes were studied by X-ray diffraction techniques. The magnetic properties of complexes 1 and 2 are reported and discussed.

Controlled Immobilization of a Palladium Complex/Laccase Hybrid into a Macrocellular Siliceous Host.

This study investigates the site-directed immobilization of a hybrid catalyst bearing a biquinoline-based-Pd(II) complex (1) and a robust laccase within cavities of a silica foam to favor veratryl alcohol oxidation. We performed the grafting of 1 at a unique surface located lysine of two laccase variants, either at closed (1⊂UNIK157 ) or opposite position (1⊂UNIK71 ) of the enzyme oxidation site. After immobilization into the cavities of silica monoliths bearing hierarchical porosity, we show that catalytic activity is dependent on the orientation and loading of each hybrid, 1⊂UNIK157 being twice as active than 1⊂UNIK71 (203 TON vs 100 TON) when operating under continuous flow. These systems can be reused 5 times, with an operational activity remaining as high as 40 %. We show that the synergy between 1 and laccase can be tuned within the foam. This work is a proof of concept for controlling the organization of a heterogeneous hybrid catalyst using a Pd/laccase/silica foam.

1-Aminocyclopropane-1-carboxylic acid oxidase: insight into cofactor binding from experimental and theoretical studies.

1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a nonheme Fe(II)-containing enzyme that is related to the 2-oxoglutarate-dependent dioxygenase family. The binding of substrates/cofactors to tomato ACCO was investigated through kinetics, tryptophan fluorescence quenching, and modeling studies. α-Aminophosphonate analogs of the substrate (1-aminocyclopropane-1-carboxylic acid, ACC), 1-aminocyclopropane-1-phosphonic acid (ACP) and (1-amino-1-methyl)ethylphosphonic acid (AMEP), were found to be competitive inhibitors versus both ACC and bicarbonate (HCO(3)(-)) ions. The measured dissociation constants for Fe(II) and ACC clearly indicate that bicarbonate ions improve both Fe(II) and ACC binding, strongly suggesting a stabilization role for this cofactor. A structural model of tomato ACCO was constructed and used for docking experiments, providing a model of possible interactions of ACC, HCO(3)(-), and ascorbate at the active site. In this model, the ACC and bicarbonate binding sites are located close together in the active pocket. HCO(3)(-) is found at hydrogen-bond distance from ACC and interacts (hydrogen bonds or electrostatic interactions) with residues K158, R244, Y162, S246, and R300 of the enzyme. The position of ascorbate is also predicted away from ACC. Individually docked at the active site, the inhibitors ACP and AMEP were found coordinating the metal ion in place of ACC with the phosphonate groups interacting with K158 and R300, thus interlocking with both ACC and bicarbonate binding sites. In conclusion, HCO(3)(-) and ACC together occupy positions similar to the position of 2-oxoglutarate in related enzymes, and through a hydrogen bond HCO(3)(-) likely plays a major role in the stabilization of the substrate in the active pocket.

Photocatalytic generation of a non-heme Fe(iii)-hydroperoxo species with O2 in water for the oxygen atom transfer reaction.

Coupling a photoredox module and a bio-inspired non-heme model to activate O2 for the oxygen atom transfer (OAT) reaction requires a vigorous investigation to shed light on the multiple competing electron transfer steps, charge accumulation and annihilation processes, and the activation of O2 at the catalytic unit. We found that the efficient oxidative quenching mechanism between a [Ru(bpy)3]2+ chromophore and a reversible electron mediator, methyl viologen (MV2+), to form the reducing species methyl viologen radical (MV˙+) can convey an electron to O2 to form the superoxide radical and reset an Fe(iii) species in a catalytic cycle to the Fe(ii) state in an aqueous solution. The formation of the Fe(iii)-hydroperoxo (FeIII-OOH) intermediate can evolve to a highly oxidized iron-oxo species to perform the OAT reaction to an alkene substrate. Such a strategy allows us to bypass the challenging task of charge accumulation at the molecular catalytic unit for the two-electron activation of O2. The FeIII-OOH catalytic precursor was trapped and characterized by EPR spectroscopy pertaining to a metal assisted catalysis. Importantly, we found that the substrate itself can act as an electron donor to reset the photooxidized chromophore in the initial state closing the photocatalytic loop and hence excluding the use of a sacrificial electron donor. Laser Flash Photolysis (LFP) studies and spectroscopic monitoring during photocatalysis lend credence to the proposed catalytic cycle.

Site directed confinement of laccases in a porous scaffold towards robustness and selectivity.

We immobilized a fungal laccase with only two spatially close lysines available for functionalization into macrocellular Si(HIPE) monoliths for the purpose of continuous flow catalysis. Immobilization (30-45 % protein immobilization yields) was obtained using a covalent bond forming reaction between the enzyme and low glutaraldehyde (0.625 % (w/w)) functionalized foams. Testing primarily HBT-mediated RB5 dye decolorization in continuous flow reactors, we show that the activity of the heterogeneous catalyst is comparable to its homogeneous counterpart. More, its operational activity remains as high as 60 % after twelve consecutive decolorization cycles as well as after one-year storage, performances remarkable for such a material. We further immobilized two variants of the laccase containing a unique lysine: one located in the vicinity of the substrate oxidation site (K157) and one at the opposite side of this oxidation site (K71) to study the effect of the proximity of the Si(HIPE) surface on enzyme activity. Comparing activities on different substrates for monoliths with differentially oriented catalysts, we show a twofold discrimination for ABTS relative to ascorbate. This study provides ground for the development of neo-functionalized materials that beyond allowing stability and reusability will become synergic partners in the catalytic process.

Modulation of laccase catalysed oxidations at the surface of magnetic nanoparticles.

We explored the coupling of laccases to magnetic nanoparticles (MNPs) with different surface chemical coating. Two laccase variants offering two opposite and precise orientations of the substrate oxidation site were immobilised onto core-shell MNPs presenting either aliphatic aldehyde, aromatic aldehyde or azide functional groups at the particles surface. Oxidation capabilities of the six-resulting laccase-MNP hybrids were compared on ABTS and coniferyl alcohol. Herein, we show that the original interfaces created differ substantially in their reactivities with an amplitude from 1 to > 4 folds depending on the nature of the substrate. Taking enzyme orientation into account in the design of surface modification represents a way to introduce selectivity in laccase catalysed reactions.

Degradation of aflatoxin B1 by a recombinant laccase from Trametes sp. C30 expressed in Saccharomyces cerevisiae: A mechanism assessment study in vitro and in vivo.

Aflatoxin B1 (AFB1) is the most harmful mycotoxin and presents risks to human health. Utilization of enzyme to degrade AFB1 is a promising strategy to overcome this problem. In this study, we evaluated the effect of recombinant laccase expressed in Saccharomyces cerevisiae on the degradation of AFB1. It was found that AFB1 could be degraded effectively by laccase up to 91%.The results of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) showed that there were four main degradation products of AFB1 including C16H22O4, C14H16N2O2, C7H12N6O and C24H30O6. Two possible degradation pathways were proposed: 1) AFB1 lost -CO continuously, and then double bonds of furan ring were broken after reactions with H2O, H+, and -NH2; 2) AFB1 occurred decarbonylation reaction after losing -CO and double bonds were broken by additional reaction with H+. Two toxicological activity sites in AFB1, including a double bond of furo-furan ring and lactone ring in the coumarin in moiety, were destroyed. The toxicity of AFB1 degradation products was evaluated on HepG2 cells and in vivo tests, and the results indicated a decrease in hepatocytes apoptosis, liver and kidney histopathological lesions, oxidative stress, and inflammation as compared to non-laccase degraded AFB1. Moreover, the AFB1 degradation products significantly decreased the cytotoxicity and hepatotoxicity. This investigation provides innovative evidence on the effectiveness of laccase expressed in Saccharomyces cerevisiae in detoxifying AFB1.

Tracking light-induced electron transfer toward O2 in a hybrid photoredox-laccase system.

Photobiocatalysis uses light to perform specific chemical transformations in a selective and efficient way. The intention is to couple a photoredox cycle with an enzyme performing multielectronic catalytic activities. Laccase, a robust multicopper oxidase, can be envisioned to use dioxygen as a clean electron sink when coupled to an oxidation photocatalyst. Here, we provide a detailed study of the coupling of a [Ru(bpy)3]2+ photosensitizer to laccase. We demonstrate that efficient laccase reduction requires an electron relay like methyl viologen. In the presence of dioxygen, electrons transiently stored in superoxide ions are scavenged by laccase to form water instead of H2O2. The net result is the photo accumulation of highly oxidizing [Ru(bpy)3]3+. This study provides ground for the use of laccase in tandem with a light-driven oxidative process and O2 as one-electron transfer relay and as four-electron substrate to be a sustainable final electron acceptor in a photocatalytic process.