Quantification of Enzymatic Biofilm Removal Using the Sauerbrey Equation: Application to the Case of Pseudomonas protegens.

A methodology for the quantitative analysis of enzymatic removal of biofilms (BF) was developed, based on a quartz crystal microbalance (QCM) under stationary conditions. This was applied to the case of Pseudomonas protegens (PP) BFs, through a series of five enzymes, whose removal activity was screened using the presented methodology. The procedure is based on the following: when BFs can be modeled as rigid materials, QCM can be used as a balance under stationary conditions for determining the BFs mass reduction by enzymatic removal. For considering a BF as a rigid model, energy dissipation effects, associated with viscoelastic properties of the BF, must be negligible. Hence, a QCM system with detection of dissipation (referred to as QCM with dissipation) was used for evaluating the energy losses, which, in fact, resulted in negligible energy losses in the case of dehydrated PP BFs, validating the application of the Sauerbrey equation for the change of mass calculations. The stationary methodology reduces operating times and simplifies data analysis in comparison to dynamic approaches based on flow setups, which requires the incorporation of dissipation effects due to the liquid media. By carrying out QCM, glycosidase-type enzymes showed BF removal higher than 80% at enzyme concentration 50 ppm, reaching removal over 90% in the cases of amylase and cellulase/xylanase enzymes. The highest removal percentage produced a reduction from about 15 to 1 µg in the BF mass. Amylase enzyme was tested from below 50 to 1 ppm, reaching around 60% of removal at 1 ppm. The obtained results were supported by other instrumental techniques such as Raman spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, atomic force microscopy, high performance anion exchange chromatography, thermogravimetric analysis, and differential scanning calorimetry. The removal quantifications obtained with QCM were compared with those obtained by well-established screening techniques (UV-vis spectrophotometry using crystal violet and agar diffusion test). The proposed methodology expands the possibility of using a quartz microbalance to perform enzymatic activity screening.

Operando detection and suppression of spurious singlet oxygen in Li-O2 batteries.

The rechargeable lithium air (oxygen) battery (Li-O2) has very high energy density, comparable to that of fossil fuels (∼3600 W h kg-1). However, the parasitic reactions of the O2 reduction products with solvent and electrolyte lead to capacity fading and poor cyclability. During the oxygen reduction reaction (ORR) in aprotic solvents, the superoxide radical anion (O2˙-) is the main one-electron reaction product, which in the presence of Li+ ions undergoes disproportionation to yield Li2O2 and O2, a fraction of which results in singlet oxygen (1O2). The very reactive 1O2 is responsible for the spurious reactions that lead to high charging overpotential and short cycle life due to solvent and electrolyte degradation. Several techniques have been used for the detection and suppression of 1O2 inside a Li-O2 battery under operation and to test the efficiency and electrochemical stability of different physical quenchers of 1O2: azide anions, 1,4-diazabicyclo[2.2.2]octane (DABCO) and triphenylamine (TPA) in different solvents (dimethyl sulfoxide (DMSO), diglyme and tetraglyme). Operando detection of 1O2 inside the battery was accomplished by following dimethylanthracene fluorescence quenching using a bifurcated optical fiber in front-face mode through a quartz window in the battery. Differential oxygen-pressure measurements during charge-discharge cycles vs. charge during battery operation showed that the number of electrons per oxygen molecule was n > 2 in the absence of physical quenchers of 1O2, due to spurious reactions, and n = 2 in the presence of physical quenchers of 1O2, proving the suppression of spurious reactions. Battery cycling at a limited specific capacity of 500 mA h gC-1 for the MWCNT cathode and 250 mA gC-1 current density, in the absence and presence of a physical quencher or a physical quencher plus the redox mediator I3-/I- (with a lithiated Nafion® membrane), showed increasing cyclability according to coulombic efficiency and cell voltage data over 100 cycles. Operando Raman studies with a quartz window at the bottom of the battery allowed detection of Li2O2 and excess I3- redox mediator during discharge and charge, respectively.

Autocatalytic Mechanism in the Anaerobic Reduction of Metmyoglobin by Sulfide Species.

The mechanism of the metal centered reduction of metmyoglobin (MbFeIII) by sulfide species (H2S/HS-) under an argon atmosphere has been studied by a combination of spectroscopic, kinetic, and computational methods. Asymmetric S-shaped time-traces for the formation of MbFeII at varying ratios of excess sulfide were observed at pH 5.3 < pH < 8.0 and 25 °C, suggesting an autocatalytic reaction mechanism. An increased rate at more alkaline pHs points to HS- as relevant reactive species for the reduction. The formation of the sulfanyl radical (HS•) in the slow initial phase was assessed using the spin-trap phenyl N-tert-butyl nitrone. This radical initiates the formation of S-S reactive species as disulfanuidyl/ disulfanudi-idyl radical anions and disulfide (HSSH•-/HSS•2- and HSS-, respectively). The autocatalysis has been ascribed to HSS-, formed after HSSH•-/HSS•2- disproportionation, which behaves as a fast reductant toward the intermediate complex MbFeIII(HS-). We propose a reaction mechanism for the sulfide-mediated reduction of metmyoglobin where only ferric heme iron initiates the oxidation of sulfide species. Beside the chemical interest, this insight into the MbFeIII/sulfide reaction under an argon atmosphere is relevant for the interpretation of biochemical aspects of ectopic myoglobins found on hypoxic tissues toward reactive sulfur species.

Electrochemical Actuation of a DyP Peroxidase: A Facile Method for Drastic Improvement of the Catalytic Performance.

Dye decolorizing peroxidases (DyP) have attracted interest for applications such as dye-containing wastewater remediation and biomass processing. So far, efforts to improve operational pH ranges, activities, and stabilities have focused on site-directed mutagenesis and directed evolution strategies. Here, we show that the performance of the DyP from Bacillus subtilis can be drastically boosted without the need for complex molecular biology procedures by simply activating the enzyme electrochemically in the absence of externally added H2O2. Under these conditions, the enzyme shows specific activities toward a variety of chemically different substrates that are significantly higher than in its canonical operation. Moreover, it presents much broader pH activity profiles with the maxima shifted toward neutral to alkaline. We also show that the enzyme can be successfully immobilized on biocompatible electrodes. When actuated electrochemically, the enzymatic electrodes have two orders of magnitude higher turnover numbers than with the standard H2O2-dependent operation and preserve about 30% of the initial electrocatalytic activity after 5 days of operation-storage cycles.

Reduction of metmyoglobin by inorganic disulfide species.

The mechanism of the metal centered reduction of metmyoglobin (MbFeIII) by inorganic disulfide species has been studied by combined spectroscopic and kinetic analyses, under argon atmosphere. The process is kinetically characterized by biexponential time traces, for variable ratios of excess disulfide to protein, in the pH interval 6.6-8.0. Using UV-vis and resonance Raman spectroscopies, we observed that MbFeIII is converted into a low spin hexacoordinated ferric complex, tentatively assigned as MbFeIII(HSS-)/MbFeIII(SS2-), in an initial fast step. The complex is slowly converted into a pentacoordinated ferrous form, assigned as MbFeII according to the resonance Raman records. The reduction is a pH-dependent process, but independent of the initial disulfide concentration, suggesting the unimolecular decomposition of the intermediate complex following a reductive homolysis. We estimated the rate of the fast formation of the complex at pH 7.4 (kon = 3.7 × 103 M-1 s-1), and a pKa2 = 7.5 for the equilibrium MbFeIII(HSS-)/MbFeIII(SS2-). Also, we estimated the rate for the slow reduction at the same pH (kred = 10-2 s-1). A reaction mechanism compliant with the experimental results is proposed. This mechanistic study provides a differential kinetic signature for the reactions of disulfide compared to sulfide species on metmyoglobin, which may be considered in other hemeprotein systems.

Electrochemical characterization of an engineered red copper protein featuring an unprecedented entropic control of the reduction potential.

Copper is a ubiquitous metal in biology that, among other functions, is implicated in enzymatic redox catalysis and in protein electron transfer (ET). When it comes to ET, copper sites are found in two main forms, mononuclear type 1 (T1) and binuclear CuA sites, which share a common cupredoxin fold. Other relevant copper sites are the so-called type 2 (T2), which are more resilient to undergo direct electrochemistry and are usually involved in catalysis. Here we report the electrochemical and spectroscopic characterization of a novel T2-like copper site engineered following the loop swapping strategy. The ligand loop sequence of the newly discovered T1 copper site from Nitrosopumilus maritimus was introduced into the CuA scaffold from Thermus thermophilus yielding a chimeric protein that shows spectroscopic features different from both parental proteins, and resemble those of red T2 copper sites, albeit with a shorter Cu-S(Cys) bond length. The novel T2 site undergoes efficient direct electrochemistry, which allows performing temperature-dependent cyclic voltammetry studies. The obtained results reveal that this chimera constitutes the first example of a copper protein with entropically controlled reduction potential, thereby contrasting the enthalpic supremacy observed for all other copper sites reported so far. The underlying bases for this entropic control are critically discussed.

Correlated electric field modulation of electron transfer parameters and the access to alternative conformations of multifunctional cytochrome c.

Cytochrome c (Cytc) is a multifunctional protein that, in its native conformation, shuttles electrons in the mitochondrial respiratory chain. Conformational transitions that involve replacement of the heme distal ligand lead to the gain of alternative peroxidase activity, which is crucial for membrane permeabilization during apoptosis. Using a time-resolved SERR spectroelectrochemical approach, we found that the key physicochemical parameters that characterize the electron transfer (ET) canonic function and those that determine the transition to alternative conformations are strongly correlated and are modulated by local electric fields (LEF) of biologically meaningful magnitude. The electron shuttling function is optimized at moderate LEFs of around 1 V nm-1. A decrease of the LEF is detrimental for ET as it rises the reorganization energy. Moreover, LEF values below and above the optimal for ET favor alternative conformations with peroxidase activity and downshifted reduction potentials. The underlying proposed mechanism is the LEF modulation of the flexibility of crucial protein segments, which produces a differential effect on the kinetic ET and conformational parameters of Cytc. These findings might be related to variations in the mitochondrial membrane potential during apoptosis, as the basis for the switch between canonic and alternative functions of Cytc. Moreover, they highlight the possible role of variable LEFs in determining the function of other moonlighting proteins through modulation of the protein dynamics.

Direct Electrochemical Generation of Catalytically Competent Oxyferryl Species of Classes I and P Dye Decolorizing Peroxidases.

This work introduces a novel way to obtain catalytically competent oxyferryl species for two different dye-decolorizing peroxidases (DyPs) in the absence of H2O2 or any other peroxide by simply applying a reductive electrochemical potential under aerobic conditions. UV-vis and resonance Raman spectroscopies show that this method yields long-lived compounds II and I for the DyPs from Bacillus subtilis (BsDyP; Class I) and Pseudomonas putida (PpDyP; Class P), respectively. Both electrochemically generated high valent intermediates are able to oxidize ABTS at both acidic and alkaline pH. Interestingly, the electrocatalytic efficiencies obtained at pH 7.6 are very similar to the values recorded for regular catalytic ABTS/H2O2 assays at the optimal pH of the enzymes, ca. 3.7. These findings pave the way for the design of DyP-based electrocatalytic reactors operable in an extended pH range without the need of harmful reagents such as H2O2.

Reactivity of inorganic sulfide species towards a pentacoordinated heme model system.

The reactivity of inorganic sulfide towards ferric bis(N-acetyl)- microperoxidase 11 in sodium dodecyl sulfate has been explored by means of visible absorption and resonance Raman spectroscopies. The reaction has been previously studied in buffered solutions at neutral pH and in the presence of excess sulfide, revealing the formation of a moderately stable hexacoordinated low spin ferric sulfide complex that yields the ferrous form in the hour's timescale. In the surfactant solution, instead, the ferrous form is rapidly formed. The spectroscopic characterization of the heme structure in the surfactant milieu revealed the stabilization of a major ferric mono-histidyl high spin heme, which may be ascribed to out of plane distortions prompting the detachment of the axially ligated water molecule, thus leading to a differential reactivity. The ferric bis(N-acetyl)- microperoxidase 11 in sodium dodecyl sulfate provides a model for pentacoordinated heme platforms with an imidazole-based ligand.

In Situ Spectroelectrochemical Investigations of Electrode-Confined Electron-Transferring Proteins and Redox Enzymes.

This perspective analyzes recent advances in the spectroelectrochemical investigation of redox proteins and enzymes immobilized on biocompatible or biomimetic electrode surfaces. Specifically, the article highlights new insights obtained by surface-enhanced resonance Raman (SERR), surface-enhanced infrared absorption (SEIRA), protein film infrared electrochemistry (PFIRE), polarization modulation infrared reflection-absorption spectroscopy (PMIRRAS), Förster resonance energy transfer (FRET), X-ray absorption spectroscopy (XAS), electron paramagnetic resonance (EPR), and differential electrochemical mass spectrometry (DMES)-based spectroelectrochemical methods on the structure, orientation, dynamics, and reaction mechanisms for a variety of immobilized species. This includes small heme and copper electron shuttling proteins, large respiratory complexes, hydrogenases, multicopper oxidases, alcohol dehydrogenases, endonucleases, NO-reductases, and dye decolorizing peroxidases, among other enzymes. Finally, I discuss the challenges and foreseeable future developments toward a better understanding of the functioning of these complex macromolecules and their exploitation in technological devices.

CuA-based chimeric T1 copper sites allow for independent modulation of reorganization energy and reduction potential.

Attaining rational modulation of thermodynamic and kinetic redox parameters of metalloproteins is a key milestone towards the (re)design of proteins with new or improved redox functions. Here we report that implantation of ligand loops from natural T1 proteins into the scaffold of a CuA protein leads to a series of distorted T1-like sites that allow for independent modulation of reduction potentials (E°') and electron transfer reorganization energies (λ). On the one hand E°' values could be fine-tuned over 120 mV without affecting λ. On the other, λ values could be modulated by more than a factor of two while affecting E°' only by a few millivolts. These results are in sharp contrast to previous studies that used T1 cupredoxin folds, thus highlighting the importance of the protein scaffold in determining such parameters.

Purple Mixed-Valent Copper A.

CuA is a binuclear copper center acting as an electron transfer hub in terminal oxidases such as cytochrome c oxidase and nitrous oxide reductase. Its unique electronic structure is intimately linked to its function and has puzzled the community of biological inorganic chemistry for decades. Here we review the insights provided by different spectroscopic techniques of CuA centers, and the different experimental approaches to tackle its study, that encompass the synthesis of model compounds as well as protein engineering efforts. The contribution of the electronic structure to the thermodynamic and kinetic of electron transfer is extensively discussed. We also describe the proposed mechanism of CuAassembly in different organisms. The recent discovery of a novel CuA site opens new perspectives to this field.

Liquid-polymer triboelectricity: chemical mechanisms in the contact electrification process.

Liquid-polymer contact electrification between sliding water drops and the surface of polytetrafluoroethylene (PTFE) was studied as a function of the pH and ionic strength of the drop as well as ambient relative humidity (RH). The PTFE surface was characterized by using SEM, water-contact-angle measurements, FTIR spectroscopy, XPS, and Raman spectroscopy. The charge acquired by the drops was calculated by detecting the transient voltage induced on a specifically designed capacitive sensor. It is shown that water drops become positively charged at pH > pHzch (pHzch being the zero charge point of the polymer) while they become negatively charged for pH < pHzch. The addition of non-hydrolysable salts (NaCl or CaCl2) to water decreases the electrical charge induced in the drop. The charge also decreases with increasing RH. These results suggest proton or hydroxyl transfer from the liquid to the hydrophobic polymer surface. A proposed thermodynamic model for the ion transfer process allows explaining the observed effects of RH, pH and ionic strength.

Unexpected electron spin density on the axial methionine ligand in CuA suggests its involvement in electron pathways.

The CuA center is a paradigm for the study of long-range biological electron transfer. This metal center is an essential cofactor for terminal oxidases like cytochrome c oxidase, the enzymatic complex responsible for cellular respiration in eukaryotes and in most bacteria. CuA acts as an electron hub by transferring electrons from reduced cytochrome c to the catalytic site of the enzyme where dioxygen reduction takes place. Different electron transfer pathways have been proposed involving a weak axial methionine ligand residue, conserved in all CuA sites. This hypothesis has been challenged by theoretical calculations indicating the lack of electron spin density in this ligand. Here we report an NMR study with selectively labeled methionine in a native CuA. NMR spectroscopy discloses the presence of net electron spin density in the methionine axial ligand in the two alternative ground states of this metal center. Similar spin delocalization observed on two second sphere mutants further supports this evidence. These data provide a novel view of the electronic structure of CuA centers and support previously neglected electron transfer pathways.

Electron transfer and conformational transitions of cytochrome c are modulated by the same dynamical features.

Cytochrome c is a prototypical multifunctional protein that is implicated in a variety of processes that are essential both for sustaining and for terminating cellular life. Typically, alternative functions other than canonical electron transport in the respiratory chain are associated to alternative conformations. In this work we apply a combined experimental and computational study of Cyt c variants to assess whether the parameters that regulate the canonical electron transport function of Cyt c are correlated with those that determine the transition to alternative conformations, using the alkaline transition as a model conformational change. The results show that pKa values of the alkaline transition correlate with the activation energies of the frictionally-controlled electron transfer reaction, and that both parameters are mainly modulated by the flexibility of the Ω-loop 70-85. Reduction potentials and non-adiabatic ET reorganization energies, on the other hand, are both modulated by the flexibilities of the Ω-loops 40-57 and 70-85. Finally, all the measured thermodynamic and kinetic parameters that characterize both types of processes exhibit systematic variations with the dynamics of the hydrogen bond between the axial ligand Met80 and the second sphere ligand Tyr67, thus highlighting the critical role of Tyr67 in controlling canonical and alternative functions of Cyt c.

pH-Induced Binding of the Axial Ligand in an Engineered CuA Site Favors the πu State.

CuA centers perform efficient long-range electron transfer. The electronic structure of native CuA sites can be described by a double-potential well with a dominant σu* ground state in fast equilibrium with a less populated πu ground state. Here, we report a CuA mutant in which a lysine was introduced in the axial position. This results in a highly unstable protein with a pH-dependent population of the two ground states. Deep analysis of the high-pH form of this variant shows the stabilization of the πu ground state due to direct binding of the Lys residue to the copper center that we attribute to deprotonation of this residue.

The alkaline transition of cytochrome c revisited: Effects of electrostatic interactions and tyrosine nitration on the reaction dynamics.

Here we investigated the effect of electrostatic interactions and of protein tyrosine nitration of mammalian cytochrome c on the dynamics of the so-called alkaline transition, a pH- and redox-triggered conformational change that implies replacement of the axial ligand Met80 by a Lys residue. Using a combination of electrochemical, time-resolved SERR spectroelectrochemical experiments and molecular dynamics simulations we showed that in all cases the reaction can be described in terms of a two steps minimal reaction mechanism consisting of deprotonation of a triggering group followed by ligand exchange. The pKaalk values of the transition are strongly modulated by these perturbations, with a drastic downshift upon nitration and an important upshift upon establishing electrostatic interactions with a negatively charged model surface. The value of pKaalk is determined by the interplay between the acidity of a triggering group and the kinetic constants for the forward and backward ligand exchange processes. Nitration of Tyr74 results in a change of the triggering group from Lys73 in WT Cyt to Tyr74 in the nitrated protein, which dominates the pKaalk downshift towards physiological values. Electrostatic interactions, on the other hand, result in strong acceleration of the backward ligand exchange reaction, which dominates the pKaalk upshift. The different physicochemical conditions found here to influence pKaalk are expected to vary depending on cellular conditions and subcellular localization of the protein, thus determining the existence of alternative conformations of Cyt in vivo.

Fine Tuning of Functional Features of the CuA Site by Loop-Directed Mutagenesis.

Here we report the spectroscopic and electrochemical characterization of three novel chimeric CuA proteins in which either one or the three loops surrounding the metal ions in the Thermus thermophilus protein have been replaced by homologous human and plant sequences while preserving the set of coordinating amino acids. These conservative modifications mimic basic differences between CuA sites from different organisms and allow for fine tuning the energy gap between alternative electronic ground states of CuA.. This results in a systematic modulation of thermodynamic and kinetic electron transfer (ET) parameters and in the selection of one of two possible redox-active molecular orbitals, which differ in the ET reorganization energy by a factor of 2. Moreover, the ET mechanism is found to be frictionally controlled, and the modifications introduced into the different chimeras do not affect the frictional activation parameter.

Dramatic Electronic Perturbations of CuA Centers via Subtle Geometric Changes.

CuA is a binuclear copper site acting as electron entry port in terminal heme-copper oxidases. In the oxidized form, CuA is a mixed valence pair whose electronic structure can be described using a potential energy surface with two minima, σu* and πu, that are variably populated at room temperature. We report that mutations in the first and second coordination spheres of the binuclear metallocofactor can be combined in an additive manner to tune the energy gap and, thus, the relative populations of the two lowest-lying states. A series of designed mutants span σu*/πu energy gaps ranging from 900 to 13 cm-1. The smallest gap corresponds to a variant with an effectively degenerate ground state. All engineered sites preserve the mixed-valence character of this metal center and the electron transfer functionality. An increase of the Cu-Cu distance less than 0.06 Å modifies the σu*/πu energy gap by almost 2 orders of magnitude, with longer distances eliciting a larger population of the πu state. This scenario offers a stark contrast to synthetic systems, as model compounds require a lengthening of 0.5 Å in the Cu-Cu distance to stabilize the πu state. These findings show that the tight control of the protein environment allows drastic perturbations in the electronic structure of CuA sites with minor geometric changes.

Engineering a bifunctional copper site in the cupredoxin fold by loop-directed mutagenesis.

Copper sites in proteins are designed to perform either electron transfer or redox catalysis. Type 1 and CuA sites are electron transfer hubs bound to a rigid protein fold that prevents binding of exogenous ligands and side reactions. Here we report the engineering of two Type 1 sites by loop-directed mutagenesis within a CuA scaffold with unique electronic structures and functional features. A copper-thioether axial bond shorter than the copper-thiolate bond is responsible for the electronic structure features, in contrast to all other natural or chimeric sites where the copper thiolate bond is short. These sites display highly unusual features, such as: (1) a high reduction potential despite a strong interaction with the axial ligand, which we attribute to changes in the hydrogen bond network and (2) the ability to bind exogenous ligands such as imidazole and azide. This strategy widens the possibility of using natural protein scaffolds with functional features not present in nature.