A Unified Charge Storage Mechanism to Rationalize the Electrochemical Behavior of Quinone-Based Organic Electrodes in Aqueous Rechargeable Batteries.

Due to their eco-sustainability and versatility, organic electrodes are promising candidates for large-scale energy storage in rechargeable aqueous batteries. This is notably the case of aqueous hybrid batteries that pair the low voltage of a zinc anode with the high voltage of a quinone-based (or analogue of quinone-based) organic cathode. However, the mechanisms governing their charge-discharge cycles remain poorly understood and are even a matter of debate and controversy. No consensus exists on the charge carrier in mild aqueous electrolytes, especially when working in an electrolyte containing a multivalent metal cation such as Zn2+. In this study, we comprehensively investigate the electrochemical reactivity of two model quinones, chloranil, and duroquinone, either diluted in solution or incorporated into carbon-based composite electrodes. We demonstrate that a common nine-member square scheme proton-coupled electron transfer mechanism allows us to fully describe and rationalize their electrochemical behavior in relation to the pH and chemical composition of the aqueous electrolyte. Additionally, we highlight the crucial role played by the pKas associated with the reduced states of quinones in determining the nature of the charge carrier that compensates for the negative charges reversibly injected in the active material. Finally, contrary to the widely reported findings for Zn/organic batteries, we unequivocally establish that the predominant solid-state charge carriers in Zn2+-based mild aqueous electrolytes are not multivalent Zn2+ cations but rather protons supplied by the weakly acidic hexaaqua metal ions (i.e., [Zn(H2O)6]2+]).

Multivalent-Ion versus Proton Insertion into Nanostructured Electrochromic WO3 from Mild Aqueous Electrolytes.

Mild aqueous electrolytes containing multivalent metal salts are currently scrutinized for the development of ecosustainable energy-related devices. However, the role of soluble multivalent metal ions in the electrochemical reactivity of transition metal oxides is a matter of debate, especially when they are performed in protic aqueous electrolytes. Here, we have compared, by means of (spectro)electrochemistry, the reversible electrochromic reduction of transparent nanostructured γ-WO3 thin films in mild aqueous electrolytes of various chemical composition and pH. This study reveals that reversible proton insertion is the only charge storage mechanism over a large pH range and that it is effective for aqueous electrolytes prepared from either organic (such as acetic acid) or inorganic (such as solvated multivalent cations) Bro̷nsted acids. By refuting charge storage mechanisms relying on the reversible insertion of multivalent metal ions, notably in aqueous electrolytes based on Al3+ ions or a mixture of Al3+ and Zn2+ ions, these fundamental results pave the way for the rational development of electrolytes and active materials for a range of aqueous-based devices, such as the emerging concept of an energy-saving smart window, which we also address in this study.

The Role of Al3+ -Based Aqueous Electrolytes in the Charge Storage Mechanism of MnOx Cathodes.

Rechargeable aqueous aluminium batteries are the subject of growing interest, however, the charge storage mechanisms at manganese oxide-based cathodes remain poorly understood. In essense, every study proposes a different mechanism. Here, an in situ spectroelectrochemical methodology is used to unambiguously demonstrate that reversible proton-coupled MnO2 -to-Mn2+ conversion is the main charge storage mechanism occurring at MnO2 cathodes for a range of slightly acidic Al3+ -based aqueous electrolytes, with the Al3+ hexaaquo complex playing the key role of proton donor. In Zn/MnO2 assemblies, this mechanism is associated with high gravimetric capacities and discharge potentials, up to 560 mAh g-1 and 1.65 V respectively, attractive efficiencies (CE > 99.5% and EE > 82%) and excellent cyclability (almost 100% capacity retention over 1 400 cycles at 2 A g-1 ). Finally, a critical analysis of the data previously published on MnOx cathodes in Al3+ -based aqueous electrolytes is conducted to conclude on a universal charge storage mechanism, i.e., the reversible electrodissolution/electrodeposition of MnO2 .

On the unsuspected role of multivalent metal ions on the charge storage of a metal oxide electrode in mild aqueous electrolytes.

Insertion mechanisms of multivalent ions in transition metal oxide cathodes are poorly understood and subject to controversy and debate, especially when performed in aqueous electrolytes. To address this issue, we have here investigated the reversible reduction of nanostructured amorphous TiO2 electrodes by spectroelectrochemistry in mild aqueous electrolytes containing either a multivalent metal salt as AlCl3 or a weak organic acid as acetic acid. Our results show that the reversible charge storage in TiO2 is thermodynamically and kinetically indistinguishable when carried out in either an Al3+- or acetic acid-based electrolyte, both leading under similar conditions of pH and concentrations to an almost identical maximal charge storage of ∼115 mA h g-1. These observations are in agreement with a mechanism where the inserting/deinserting cation is the proton and not the multivalent metal cation. Analysis of the data also demonstrates that the proton source is the Brønsted weak acid present in the aqueous electrolyte, i.e. either the acetic acid or the aquo metal ion complex generated from solvation of Al3+ (i.e. [Al(H2O)6]3+). Such a proton-coupled charge storage mechanism is also found to occur with other multivalent metal ions such as Zn2+ and Mn2+, albeit with a lower efficiency than Al3+, an effect we have attributed to the lower acidity of [Zn(H2O)6]2+ and [Mn(H2O)6]2+. These findings are of fundamental importance because they shed new light on previous studies assuming reversible Al3+-insertion into metal oxides, and, more generally, they highlight the unsuspected proton donor role played by multivalent metal cations commonly involved in rechargeable aqueous batteries.

Mn-Mimochrome VI*a: An Artificial Metalloenzyme With Peroxygenase Activity.

Manganese-porphyrins are important tools in catalysis, due to their capability to promote a wide variety of synthetically valuable transformations. Despite their great reactivity, the difficulties to control the reaction selectivity and to protect the catalyst from self-degradation hamper their practical application. Compared to small-molecule porphyrin complexes, metalloenzymes display remarkable features, because the reactivity of the metal center is finely modulated by a complex interplay of interactions within the protein matrix. In the effort to combine the catalytic potential of manganese porphyrins with the unique properties of biological catalysts, artificial metalloenzymes have been reported, mainly by incorporation of manganese-porphyrins into native protein scaffolds. Here we describe the spectroscopic and catalytic properties of Mn-Mimochrome VI*a (Mn-MC6*a), a mini-protein with a manganese deuteroporphyrin active site within a scaffold of two synthetic peptides covalently bound to the porphyrin. Mn-MC6*a is an efficient catalyst endowed with peroxygenase activity. The UV-vis absorption spectrum of Mn-MC6*a resembles that of Mn-reconstituted horseradish peroxidase (Mn-HRP), both in the resting and high-valent oxidized states. Remarkably, Mn-MC6*a shows a higher reactivity compared to Mn-HRP, because higher yields and chemoselectivity were observed in thioether oxidation. Experimental evidences also provided indications on the nature of the high-valent reactive intermediate and on the sulfoxidation mechanism.

Unraveling the charge transfer/electron transport in mesoporous semiconductive TiO2 films by voltabsorptometry.

In this work, we demonstrate that chronoabsorptometry and more specifically cyclic voltabsorptometry are particularly well suited techniques for acquiring a comprehensive understanding of the dynamics of electron transfer/charge transport within a transparent mesoporous semiconductive metal oxide film loaded with a redox-active dye. This is illustrated with the quantitative analysis of the spectroelectrochemical responses of two distinct heme-based redox probes adsorbed in highly-ordered mesoporous TiO2 thin films (prepared from evaporation-induced self-assembly, EISA). On the basis of a finite linear diffusion-reaction model as well as the establishment of the analytical expressions governing the limiting cases, it was possible to quantitatively analyse, predict and interpret the unusual voltabsorptometric responses of the adsorbed redox species as a function of the potential applied to the semiconductive film (i.e., as a function of the transition from an insulating to a conductive state or vice versa). In particular, we were able to accurately determine the interfacial charge transfer rates between the adsorbed redox species and the porous semiconductor. Another important and unexpected finding, inferred from the voltabsorptograms, is an interfacial electron transfer process predominantly governed by the extended conduction band states of the EISA TiO2 film and not by the localized traps in the bandgap. This is a significant result that contrasts those previously observed for dye-sensitized solar cells formed of randomly sintered TiO2 nanoparticles, a behaviour that was ascribed to a particularly low density of localized surface states in EISA TiO2. The present methodology also provides a unique and straightforward access to an activation-driving force relationship according to the Marcus theory, thus opening new opportunities not only to investigate the driving-force effects on electron recombination dynamics in dye-sensitized solar cells but also to study the electron transfer/transport mechanisms in heterogeneous photoelectrocatalytic systems combining nanostructured semiconductor electrodes and heterogeneous redox-active catalysts.

Efficient chemisorption of organophosphorous redox probes on indium tin oxide surfaces under mild conditions.

We report a mild and straightforward one-step chemical surface functionalization of indium tin oxide (ITO) electrodes by redox-active molecules bearing an organophosphoryl anchoring group (i.e., alkyl phosphate or alkyl phosphonate group). The method takes advantage of simple passive adsorption in an aqueous solution at room temperature. We show that organophosphorus compounds can adsorb much more strongly and stably on an ITO surface than analogous redox-active molecules bearing a carboxylate or a boronate moiety. We provide evidence, through quantitative electrochemical characterization (i.e., by cyclic voltammetry) of the adsorbed organophosphoryl redox-active molecules, of the occurrence of three different adsorbate fractions on ITO, exhibiting different stabilities on the surface. Among these three fractions, one is observed to be strongly chemisorbed, exhibiting high stability and resistance to desorption/hydrolysis in a free-redox probe aqueous buffer. We attribute this remarkable stability to the formation of chemical bonds between the organophosphorus anchoring group and the metal oxide surface, likely occurring through a heterocondensation reaction in water. From XPS analysis, we also demonstrate that the surface coverage of the chemisorbed molecules is highly affected by the degree of surface hydroxylation, a parameter that can be tuned by simply preconditioning the freshly cleaned ITO surfaces in water. The lower the relative surface hydroxide density on ITO, the higher was the surface coverage of the chemisorbed species. This behavior is in line with a chemisorption mechanism involving coordination of a deprotonated phosphoryl oxygen atom to the non-hydroxylated acidic metal sites of ITO.

ATP binding turns plant cryptochrome into an efficient natural photoswitch.

Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH(·) radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD·(-), from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396(-). Its negative charge could trigger conformational changes necessary for signal transduction.

Substrate interaction dynamics and oxygen control in the active site of thymidylate synthase ThyX.

Thymidylate synthase ThyX, required for DNA synthesis in many pathogenic bacteria, is considered a promising antimicrobial target. It binds FAD and three substrates, producing dTMP (2'-deoxythymidine-5'-monophosphate) from dUMP (2'-deoxyuridine-5'-monophosphate). However, ThyX proteins also act as NADPH oxidase by reacting directly with O2. In the present study we investigated the dynamic interplay between the substrates and their role in competing with this wasteful and potentially harmful oxidase reaction in catalytically efficient ThyX from Paramecium bursaria Chlorella virus-1. dUMP binding accelerates the O2-insensitive half-reaction between NADPH and FAD by over four orders of magnitude to ~30 s-1. Thus, although dUMP does not have a direct role in FAD reduction, any turnover with molecular O2 requires its presence. Inversely, NADPH accommodation accelerates dUMP binding ~3-fold and apparently precedes dUMP binding under physiological conditions. In the oxidative half-reaction, excess CH2H4folate (N5,N10-methylene-5,6,7,8-tetrahydrofolate) was found to re-oxidize FADH2 within 1 ms, thus very efficiently competing with FADH2 oxidation by O2 (1.5 s-1 under aerobic conditions). The resulting reaction scheme points out how the interplay between the fast reactions with the native substrates, although not rate-limiting for overall catalysis, avoids NADPH oxidase activity in aerobic micro-organisms, including many pathogens. These observations also explain why ThyX proteins are also present in aerobic micro-organisms.

Toward stable electron paramagnetic resonance oximetry probes: synthesis, characterization, and metabolic evaluation of new ester derivatives of a tris-(para-carboxyltetrathiaaryl)methyl (TAM) radical.

Tris(p-carboxyltetrathiaaryl)methyl (TAM) radicals, such as 1a ("Finland" radical), are useful EPR probes for oximetry. However, they are rapidly metabolized by liver microsomes in the presence of NADPH, with the formation of diamagnetic quinone-methide metabolites resulting from an oxidative decarboxylation of one of their carboxylate substituents. In an effort to obtain TAM derivatives potentially more metabolically stable in vivo, we have synthesized four new TAM radicals in which the carboxylate substituents of 1a have been replaced with esters groups bearing various alkyl chains designed to render them water-soluble. The new compounds were completely characterized by UV-vis and EPR spectroscopies, high resolution mass spectrometry (HRMS), and electrochemistry. Two of them were water-soluble enough to undergo detailed microsomal metabolic studies in comparison with 1a. They were found to be stable in the presence of the esterases present in rat liver microsomes and cytosol, and, contrary to 1a, stable to oxidation in the presence of NADPH-supplemented microsomes. A careful study of their possible microsomal reduction under anaerobic or aerobic conditions showed that they were more easily reduced than 1a, in agreement with their higher reduction potentials. They were reduced into the corresponding anions not only under anaerobic conditions but also in the presence of dioxygen. These anions were much more stable than that of 1a and could be characterized by UV-vis spectroscopy, MS, and at the level of their protonated product. However, they were oxidized by O2, giving back to the starting ester radicals and catalyzing a futile cycle of O2 reduction. Such reactions should be considered in the design of future stable EPR probes for oximetry in vivo.

Spectroelectrochemical characterization of small hemoproteins adsorbed within nanostructured mesoporous ITO electrodes.

3D nanostructured transparent indium tin oxide (ITO) electrodes prepared by glancing angle deposition (GLAD) were used for the spectroelectrochemical characterization of cytochrome c (Cyt c) and neuroglobin (Nb). These small hemoproteins, involved as electron-transfer partners in the prevention of apoptosis, are oppositely charged at physiological pH and can each be adsorbed within the ITO network under different pH conditions. The resulting modified electrodes were investigated by UV-visible absorption spectroscopy coupled with cyclic voltammetry. By using nondenaturating adsorption conditions, we demonstrate that both proteins are capable of direct electron transfer to the conductive ITO surface, sharing apparent standard potentials similar to those reported in solution. Preservation of the 3D protein structure upon adsorption was confirmed by resonance Raman (rR) spectroscopy. Analysis of the derivative cyclic voltabsorptograms (DCVA) monitored either in the Soret or the Q bands at scan rates up to 1 V s(-1) allowed us to investigate direct interfacial electron transfer kinetics. From the DCVA shape and scan rate dependences, we conclude that the interaction of Cyt c with the ITO surface is more specific than Nb, suggesting an oriented adsorption of Cyt c and a random adsorption of Nb on the ITO surface. At the same time, Cyt c appears more sensitive to the experimental adsorption conditions, and complete denaturation of Cyt c may occur as evidenced from cross-correlation of rR spectroscopy and spectroelectrochemistry.

Unraveling the mechanism of catalytic reduction of O2 by microperoxidase-11 adsorbed within a transparent 3D-nanoporous ITO film.

Nanoporous films of indium tin oxide (ITO), with thicknesses ranging from 250 nm to 2 µm, were prepared by Glancing Angle Deposition (GLAD) and used as highly sensitive transparent 3D-electrodes for quantitatively interrogating, by time-resolved spectroelectrochemistry, the reactivity of microperoxidase-11 (MP-11) adsorbed within such films. The capacitive current densities of these 3D-electrodes as well as the amount of adsorbed MP-11 were shown to be linearly correlated to the GLAD ITO film thickness, indicating a homogeneous distribution of MP-11 across the film as well as homogeneous film porosity. Under saturating adsorption conditions, MP-11 film concentration as high as 60 mM was reached. This is equivalent to a stack of 110 monolayers of MP-11 per micrometer film thickness. This high MP-11 film loading combined with the excellent ITO film conductivity has allowed the simultaneous characterization of the heterogeneous one-electron transfer dynamics of the MP-11 Fe(III)/Fe(II) redox couple by cyclic voltammetry and cyclic voltabsorptometry, up to a scan rate of few volts per second with a satisfactory single-scan signal-to-noise ratio. The potency of the method to unravel complex redox coupled chemical reactions was also demonstrated with the catalytic reduction of oxygen by MP-11. In the presence of O(2), cross-correlation of electrochemical and spectroscopic data has allowed us to determine the key kinetics and thermodynamics parameters of the redox catalysis that otherwise could not be easily extracted using conventional protein film voltammetry. On the basis of numerical simulations of cyclic voltammograms and voltabsorptograms and within the framework of different plausible catalytic reaction schemes including appropriate approximations, it was shown possible to discriminate between different possible catalytic pathways and to identify the relevant catalytic cycle. In addition, from the best fits of simulations to the experimental voltammograms and voltabsorptograms, the partition coefficient of O(2) for the ITO film as well as the values of two kinetic rate constants could be extracted. It was finally concluded that the catalytic reduction of O(2) by MP-11 adsorbed within nanoporous ITO films occurs via a 2-electron mechanism with the formation of an intermediate Fe(III)-OOH adduct characterized by a decay rate of 11 s(-1). The spectroelectroanalytical strategy presented here opens new opportunities for characterizing complex redox-coupled chemical reactions not only with redox proteins, but also with redox biomimetic systems and catalysts. It might also be of great interest for the development and optimization of new spectroelectrochemical sensors and biosensors, or eventually new photoelectrocatalytic systems or biofuel cells.

Time-resolved UV-visible spectroelectrochemistry using transparent 3D-mesoporous nanocrystalline ITO electrodes.

Efficient and rapid adsorption of microperoxidase 11 within a highly porous ITO thin film (200 nm) prepared by glancing angle deposition was achieved. Adsorbed redox molecules were reversibly and rapidly reduced throughout the 3D-conductive matrix in ca. 50 ms, allowing the heterogeneous electron transfer rate to be determined by derivative cyclic voltabsorptometry.

Electrochemical and homogeneous electron transfers to the Alzheimer amyloid-beta copper complex follow a preorganization mechanism.

Deciphering the electron transfer reactivity characteristics of amyloid ß-peptide copper complexes is an important task in connection with the role they are assumed to play in Alzheimer's disease. A systematic analysis of this question with the example of the amyloid ß-peptide copper complex by means of its electrochemical current-potential responses and of its homogenous reactions with electrogenerated fast electron exchanging osmium complexes revealed a quite peculiar mechanism: The reaction proceeds through a small fraction of the complex molecules in which the peptide complex is "preorganized" so as the distances and angles in the coordination sphere to vary minimally upon electron transfer, thus involving a remarkably small reorganization energy (0.3 eV). This preorganization mechanism and its consequences on the reactivity should be taken into account for reactions involving dioxygen and hydrogen peroxide that are considered to be important in Alzheimer's disease through the production of harmful reactive oxygen species.

Role of arginine guanidinium moiety in nitric-oxide synthase mechanism of oxygen activation.

Nitric-oxide synthases (NOS) are highly regulated heme-thiolate enzymes that catalyze two oxidation reactions that sequentially convert the substrate L-Arg first to N(omega)-hydroxyl-L-arginine and then to L-citrulline and nitric oxide. Despite numerous investigations, the detailed molecular mechanism of NOS remains elusive and debatable. Much of the dispute in the various proposed mechanisms resides in the uncertainty concerning the number and sources of proton transfers. Although specific protonation events are key features in determining the specificity and efficiency of the two catalytic steps, little is known about the role and properties of protons from the substrate, cofactors, and H-bond network in the vicinity of the heme active site. In this study, we have investigated the role of the acidic proton from the L-Arg guanidinium moiety on the stability and reactivity of the ferrous heme-oxy complex intermediate by exploiting a series of L-Arg analogues exhibiting a wide range of guanidinium pK(a) values. Using electrochemical and vibrational spectroscopic techniques, we have analyzed the effects of the analogues on the heme, including characteristics of its proximal ligand, heme conformation, redox potential, and electrostatic properties of its distal environment. Our results indicate that the substrate guanidinium pK(a) value significantly affects the H-bond network near the heme distal pocket. Our results lead us to propose a new structural model where the properties of the guanidinium moiety finely control the proton transfer events in NOS and tune its oxidative chemistry. This model may account for the discrepancies found in previously proposed mechanisms of NOS oxidation processes.

Highly ordered transparent mesoporous TiO2 thin films: an attractive matrix for efficient immobilization and spectroelectrochemical characterization of cytochrome c.

We demonstrate remarkably fast incorporation and high loading of cytochrome c within thin films of periodically ordered nanocrystalline TiO(2) deposited on transparent electrodes. The immobilized cytochrome c is not denaturated and it can be reversibly reduced without mediator over the time scale of a few seconds as evidenced by spectroelectrochemistry.

Importance of dynamical processes in the coordination chemistry and redox conversion of copper amyloid-beta complexes.

Interaction of Cu ions with the amyloid-beta (Abeta) peptide is linked to the development of Alzheimer's disease; hence, determining the coordination of Cu(I) and Cu(II) ions to Abeta and the pathway of the Cu(I)(Abeta)/Cu(II)(Abeta) redox conversion is of great interest. In the present report, we use the room temperature X-ray absorption near edge structure to show that the binding sites of the Cu(I) and Cu(II) complexes are similar to those previously determined from frozen-solution studies. More precisely, the Cu(I) is coordinated by the imidazole groups of two histidine residues in a linear fashion. However, an NMR study unravels the involvement of all three histidine residues in the Cu(I) binding due to dynamical exchange between several set of ligands. The presence of an equilibrium is also responsible for the complex redox process observed by cyclic voltammetry and evidenced by a concentration-dependent electrochemical response.

Characterization of the electron transfer of a ferrocene redox probe and a histidine-tagged hemoprotein specifically bound to a nitrilotriacetic-terminated self-assembled monolayer.

We report the selective, controlled binding of a model redox probe, 1,1'-bis(N-imidazolylmethyl)ferrocene (Fc-Im2), and a small redox hemoprotein, histidine-tagged recombinant human neuroglobin (hNb), at the surface of metal electrodes (gold and SER-active silver) modified by a self-assembled monolayer (SAM) of a nitrilotriacetic (NTA)-terminated thiol. The resulting SAMs were characterized by cyclic voltammetry and surface-enhanced resonance Raman (SERR) spectroscopy coupled to electrochemistry. Once specifically bounded to the Ni(II)-NTA-modified gold electrode, nearly ideal cyclic voltammetric behavior with relatively fast electron-transfer (ET) communication through the SAM was determined for the Fc-Im2 redox probe. However, no direct electron transfer could be evidenced for the hNb redox protein under the same conditions. This outcome was different from the result obtained during SERR experiments coupled to electrochemistry in which a direct electrochemical conversion of hNb immobilized on a Ni(II)-NTA-modified SER-active Ag electrode was observed. The SERR spectra of the immobilized hNb was the same as the resonance Raman spectra of the protein in homogeneous solution, allowing us to conclude that the native structure of hNb was retained upon immobilization and that the direct ET was not the result of some partial or complete protein denaturation. The long-range ET rate constant (kET) through the SAM was determined by time-resolved SERR spectroscopy. A value of kET=0.12 s(-1) was obtained, which is within the predicted range of a fully nonadiabatic ET through a SAM thickness of approximately 26 A and close to the values previously determined for analogous small redox proteins at similar long-range ET distances. A SERR spectroelectrochemical titration of the immobilized hNb was also carried out, showing both an apparent standard potential (E0') negatively shifted by 100 mV compared with hNb in solution and a gentle slope in the titration curve. These results suggest a range of chemical environments in the surroundings of the redox protein and a variety of interactions with the NTA-terminated SAM. The influence of protein immobilization on E0' is discussed together with the long-range ET rate constant and molecular orientation of the surface-immobilized hNb.

What makes the difference between a cryptochrome and DNA photolyase? A spectroelectrochemical comparison of the flavin redox transitions.

Cryptochromes and DNA photolyases are highly homologous flavoproteins that accomplish completely different tasks. While plant cryptochrome1 functions as blue light photoreceptor that triggers various morphogenic reactions, photolyases repair UV-induced DNA damages. Both enzymes share the photoactive cofactor, noncovalently bound FAD. For photolyase, the reaction mechanism involves electron transfer to the substrate from the excited-state of fully reduced flavin. For cryptochrome, photoexcitation of the oxidized flavin leads to formation of the semireduced radical FADH(*). Key parameters for the redox state of the flavin in the cell are the midpoint potentials E(1) and E(2) for the oxidized/semireduced and semireduced/fully reduced transitions, respectively. A link between cryptochrome function and its cofactor's redox states has been suggested early on, but no reliable determinations of midpoint potentials have been available. Here we report spectroelectrochemical titrations of cryptochrome1 from Arabidopsis thaliana and photolyases from both E. coli and Anacystis nidulans at pH 7.4. For the cryptochrome, we obtained E(1) approximately E(2) approximately -160 mV vs NHE, strongly deviating from the photolyases where FADH(*) could not be oxidized up to 400 mV, and E(2) approximately -40 mV. Functional and evolutionary implications are discussed, highlighting the role of an asparagine-to-aspartate replacement close to N5 of the flavin.

Electrochemical functionalization of carbon surfaces by aromatic azide or alkyne molecules: a versatile platform for click chemistry.

The electrochemical reduction of phenylazide or phenylacetylene diazonium salts leads to the grafting of azido or ethynyl groups onto the surface of carbon electrodes. In the presence of copper(I) catalyst, these azide- or alkyne-modified surfaces react efficiently and rapidly with compounds bearing an acetylene or azide function, thus forming a covalent 1,2,3-triazole linkage by means of click chemistry. This was illustrated with the surface coupling of ferrocenes functionalized with an ethynyl or azido group and the biomolecule biotin terminated by an acetylene group.