Bimetallic Mn, Fe, Co, and Ni Sites in a Four-Helix Bundle Protein: Metal Binding, Structure, and Peroxide Activation.

Bimetallic active sites in enzymes catalyze small-molecule conversions that are among the top 10 challenges in chemistry. As different metal cofactors are typically incorporated in varying protein scaffolds, it is demanding to disentangle the individual contributions of the metal and the protein matrix to the activity. Here, we compared the structure, properties, and hydrogen peroxide reactivity of four homobimetallic cofactors (Mn(II)2, Fe(II)2, Co(II)2, Ni(II)2) that were reconstituted into a four-helix bundle protein. Reconstituted proteins were studied in solution and in crystals. All metals bind with high affinity and yield similar cofactor structures. Cofactor variants react with H2O2 but differ in their turnover rates, accumulated oxidation states, and trapped peroxide-bound intermediates. Varying the metal composition thus creates opportunities to tune the reactivity of the bimetallic cofactor and to study and functionalize reactive species.

Electrochemical Biosensors Employing Natural and Artificial Heme Peroxidases on Semiconductors.

Heme peroxidases are widely used as biological recognition elements in electrochemical biosensors for hydrogen peroxide and phenolic compounds. Various nature-derived and fully synthetic heme peroxidase mimics have been designed and their potential for replacing the natural enzymes in biosensors has been investigated. The use of semiconducting materials as transducers can thereby offer new opportunities with respect to catalyst immobilization, reaction stimulation, or read-out. This review focuses on approaches for the construction of electrochemical biosensors employing natural heme peroxidases as well as various mimics immobilized on semiconducting electrode surfaces. It will outline important advances made so far as well as the novel applications resulting thereof.

Modulating the Molybdenum Coordination Sphere of Escherichia coli Trimethylamine N-Oxide Reductase.

The well-studied enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. We report on a system for the in vitro reconstitution of TorA with molybdenum cofactors (Moco) from different sources. Higher TMAO reductase activities for TorA were obtained when using Moco sources containing a sulfido ligand at the molybdenum atom. For the first time, we were able to isolate functional bis-MGD from Rhodobacter capsulatus formate dehydrogenase (FDH), which remained intact in its isolated state and after insertion into apo-TorA yielded a highly active enzyme. Combined characterizations of the reconstituted TorA enzymes by electron paramagnetic resonance spectroscopy and direct electrochemistry emphasize that TorA activity can be modified by changes in the Mo coordination sphere. The combination of these results together with studies of amino acid exchanges at the active site led us to propose a novel model for binding of the substrate to the molybdenum atom of TorA.

Transient Catalytic Voltammetry of Sulfite Oxidase Reveals Rate Limiting Conformational Changes.

Sulfite oxidases are metalloenzymes that oxidize sulfite to sulfate at a molybdenum active site. In vertebrate sulfite oxidases, the electrons generated at the Mo center are transferred to an external electron acceptor via a heme domain, which can adopt two conformations: a "closed" conformation, suitable for internal electron transfer, and an "open" conformation suitable for intermolecular electron transfer. This conformational change is an integral part of the catalytic cycle. Sulfite oxidases have been wired to electrode surfaces, but their immobilization leads to a significant decrease in their catalytic activity, raising the question of the occurrence of the conformational change when the enzyme is on an electrode. We recorded and quantitatively modeled for the first time the transient response of the catalytic cycle of human sulfite oxidase immobilized on an electrode. We show that conformational changes still occur on the electrode, but at a lower rate than in solution, which is the reason for the decrease in activity of sulfite oxidases upon immobilization.

The 1,6,7,12-Tetraazaperylene Bridging Ligand as an Electron Reservoir and Its Disulfonato Derivative as Redox Mediator in an Enzyme-Electrode Process.

The homodinuclear ruthenium(II) complex [{Ru(l-N4 Me2 )}2 (µ-tape)](PF6 )4 {[1](PF6 )4 } (l-N4 Me2 =N,N'-dimethyl-2,11-diaza[3.3](2,6)-pyridinophane, tape=1,6,7,12-tetraazaperylene) can store one or two electrons in the energetically low-lying π* orbital of the bridging ligand tape. The corresponding singly and doubly reduced complexes [{Ru(l-N4 Me2 )}2 (µ-tape.- )](PF6 )3 {[2](PF6 )3 } and [{Ru(l-N4 Me2 )}2 (µ-tape2- )](PF6 )2 {[3](PF6 )2 }, respectively, were electrochemically generated, successfully isolated and fully characterized by single-crystal X-ray crystallography, spectroscopic methods and magnetic susceptibility measurements. The singly reduced complex [2](PF6 )3 contains the π-radical tape.- and the doubly reduced [3](PF6 )2 the diamagnetic dianion tape2- as bridging ligand, respectively. Nucleophilic aromatic substitution at the bridging tape in [1]4+ by two sulfite units gave the complex [{Ru(l-N4 Me2 )}2 {µ-tape-(SO3 )2 }]2+ ([4]2+ ). Complex dication [4]2+ was exploited as a redox mediator between an anaerobic homogenous reaction solution of an enzyme system (sulfite/sulfite oxidase) and the electrode via participation of the low-energy π*-orbital of the disulfonato-substituted bridging ligand tape-(SO3 )22- (Ered1 =-0.1 V versus Ag/AgCl/1 m KCl in water).

Interaction of Flavin-Dependent Fructose Dehydrogenase with Cytochrome c as Basis for the Construction of Biomacromolecular Architectures on Electrodes.

The creation of electron transfer (ET) chains based on the defined arrangement of enzymes and redox proteins on electrode surfaces represents an interesting approach within the field of bioelectrocatalysis. In this study, we investigated the ET reaction of the flavin-dependent enzyme fructose dehydrogenase (FDH) with the redox protein cytochrome c (cyt c). Two different pH optima were found for the reaction in acidic and neutral solutions. When cyt c was adsorbed on an electrode surface while the enzyme remained in solution, ET proceeded efficiently in media of neutral pH. Interprotein ET was also observed in acidic media; however, it appeared to be less efficient. These findings suggest that two different ET pathways between the enzyme and cyt c may occur. Moreover, cyt c and FDH were immobilized in multiple layers on an electrode surface by means of another biomacromolecule: DNA (double stranded) using the layer-by-layer technique. The biprotein multilayer architecture showed a catalytic response in dependence on the fructose concentration, indicating that the ET reaction between both proteins is feasible even in the immobilized state. The electrode showed a defined response to fructose and a good storage stability. Our results contribute to the better understanding of the ET reaction between FDH and cyt c and provide the basis for the creation of all-biomolecule based fructose sensors the sensitivity of which can be controlled by the layer preparation.

Mediated bioelectrochemical system for biosensing the cell viability of Staphylococcus aureus.

Staphylococcus aureus is one of the most dangerous human pathogens and is the cause of numerous illnesses ranging from moderate skin infections to life-threatening diseases. Despite advances made in identifying microorganisms, rapid detection methods for the viability of bacteria are still missing. Here, we report a rapid electrochemical assay for cell viability combining the use of double redox mediators and multiwall carbon nanotubes-screen printed electrodes (MWCNTs-SPE), ferricyanide (FCN) and 2,6-dichlorophenolindophenol (DCIP), which served as electron shuttle to enable the bacterial-electrode communications. The current originating from the metabolically active cells was recorded for probing the activity of the intracellular redox centers. Blocking of the respiratory chain pathways with electron transfer inhibitors demonstrated the involvement of the electron transport chain in the reaction. A good correlation between the number of the metabolically active cells and the current was obtained. The proposed assay has been exploited for monitoring cell proliferation of S. aureus during the growth. The sensitivity of the detection method reached 0.1 OD600. Therefore, the technique described is promising for estimating the cell number, measuring the cell viability, and probing intracellular redox center(s).

Molecularly Imprinted Electropolymer for a Hexameric Heme Protein with Direct Electron Transfer and Peroxide Electrocatalysis.

For the first time a molecularly imprinted polymer (MIP) with direct electron transfer (DET) and bioelectrocatalytic activity of the target protein is presented. Thin films of MIPs for the recognition of a hexameric tyrosine-coordinated heme protein (HTHP) have been prepared by electropolymerization of scopoletin after oriented assembly of HTHP on a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold electrodes. Cavities which should resemble the shape and size of HTHP were formed by template removal. Rebinding of the target protein sums up the recognition by non-covalent interactions between the protein and the MIP with the electrostatic attraction of the protein by the SAM. HTHP bound to the MIP exhibits quasi-reversible DET which is reflected by a pair of well pronounced redox peaks in the cyclic voltammograms (CVs) with a formal potential of -184.4 ± 13.7 mV vs. Ag/AgCl (1 M KCl) at pH 8.0 and it was able to catalyze the cathodic reduction of peroxide. At saturation the MIP films show a 12-fold higher electroactive surface concentration of HTHP than the non-imprinted polymer (NIP).

Surface-tuned electron transfer and electrocatalysis of hexameric tyrosine-coordinated heme protein.

Molecular modeling, electrochemical methods, and quartz crystal microbalance were used to characterize immobilized hexameric tyrosine-coordinated heme protein (HTHP) on bare carbon or on gold electrodes modified with positively and negatively charged self-assembled monolayers (SAMs), respectively. HTHP binds to the positively charged surface but no direct electron transfer (DET) is found due to the long distance of the active sites from the electrode surfaces. At carboxyl-terminated surfaces, the neutrally charged bottom of HTHP can bind to the SAM. For this "disc" orientation all six hemes are close to the electrode and their direct electron transfer should be efficient. HTHP on all negatively charged SAMs showed a quasi-reversible redox behavior with rate constant ks values between 0.93 and 2.86 s(-1) and apparent formal potentials ${E{{0{^{\prime }}\hfill \atop {\rm app}\hfill}}}$ between -131.1 and -249.1 mV. On the MUA/MU-modified electrode, the maximum surface concentration corresponds to a complete monolayer of the hexameric HTHP in the disc orientation. HTHP electrostatically immobilized on negatively charged SAMs shows electrocatalysis of peroxide reduction and enzymatic oxidation of NADH.

Spectroscopic Observation of Calcium-Induced Reorientation of Cellobiose Dehydrogenase Immobilized on Electrodes and its Effect on Electrocatalytic Activity.

Cellobiose dehydrogenase catalyzes the oxidation of various carbohydrates and is considered as a possible anode catalyst in biofuel cells. It has been shown that the catalytic performance of this enzyme immobilized on electrodes can be increased by presence of calcium ions. To get insight into the Ca(2+) -induced changes in the immobilized enzyme we employ surface-enhanced vibrational (SERR and SEIRA) spectroscopy together with electrochemistry. Upon addition of Ca(2+) ions electrochemical measurements show a shift of the catalytic turnover signal to more negative potentials while SERR measurements reveal an offset between the potential of heme reduction and catalytic current. Comparing SERR and SEIRA data we propose that binding of Ca(2+) to the heme induces protein reorientation in a way that the electron transfer pathway of the catalytic FAD center to the electrode can bypass the heme cofactor, resulting in catalytic activity at more negative potentials.

Characterization of the enhanced peroxidatic activity of amyloid ß peptide-hemin complexes towards neurotransmitters.

Binding of heme to the amyloid peptides Aß40/42 is thought to be an initial step in the development of symptoms in the early stages of Alzheimer's disease by enhancing the intrinsic peroxidatic activity of heme. We found considerably higher acceleration of the reaction for the physiologically relevant neurotransmitters dopamine and serotonin than reported earlier for the artificial substrate 3,3',5,5'-tetramethylbenzidine (TMB). Thus, the binding of hemin to Aß peptides might play an even more crucial role in the early stages of Alzheimer's disease than deduced from these earlier results. To mimic complex formation, a new surface architecture has been developed: The interaction between the truncated amyloid peptide Aß1-16 and hemin immobilized on an aminohexanethiol spacer on a gold electrode has been analyzed by cyclic voltammetry. The resulting complex has a redox pair with a 25 mV more cathodic formal potential than hemin alone.

The aromatic peroxygenase from Marasmius rutola--a new enzyme for biosensor applications.

The aromatic peroxygenase (APO; EC 1.11.2.1) from the agraric basidomycete Marasmius rotula (MroAPO) immobilized at the chitosan-capped gold-nanoparticle-modified glassy carbon electrode displayed a pair of redox peaks with a midpoint potential of -278.5 mV vs. AgCl/AgCl (1 M KCl) for the Fe(2+)/Fe(3+) redox couple of the heme-thiolate-containing protein. MroAPO oxidizes aromatic substrates such as aniline, p-aminophenol, hydroquinone, resorcinol, catechol, and paracetamol by means of hydrogen peroxide. The substrate spectrum overlaps with those of cytochrome P450s and plant peroxidases which are relevant in environmental analysis and drug monitoring. In M. rotula peroxygenase-based enzyme electrodes, the signal is generated by the reduction of electrode-active reaction products (e.g., p-benzoquinone and p-quinoneimine) with electro-enzymatic recycling of the analyte. In these enzyme electrodes, the signal reflects the conversion of all substrates thus representing an overall parameter in complex media. The performance of these sensors and their further development are discussed.

How an ACE2 mimicking epitope-MIP nanofilm recognizes template-related peptides and the receptor binding domain of SARS-CoV-2.

Here we aim to gain a mechanistic understanding of the formation of epitope-imprinted polymer nanofilms using a non-terminal peptide sequence, i.e. the peptide GFNCYFP (G485 to P491) of the SARS-CoV-2 receptor binding domain (RBD). This epitope is chemisorbed on the gold surface through the central cysteine 488 followed by the electrosynthesis of a ∼5 nm thick polyscopoletin film around the surface confined templates. The interaction of peptides and the parent RBD and spike protein with the imprinted polyscopoletin nanofilm was followed by electrochemical redox marker gating, surface enhanced infrared absorption spectroscopy and conductive AFM. Because the use of non-terminal epitopes is especially intricate, here we characterize the binding pockets through their interaction with 5 peptides rationally derived from the template sequence, i.e. implementing central single amino acid mismatch as well as elongations and truncations at its C- and N- termini. Already a single amino acid mismatch, i.e. the central Cys488 substituted by a serine, results in ca. 15-fold lower affinity. Further truncation of the peptides to tetrapeptide (EGFN) and hexapeptide (YFPLQS) results also in a significantly lower affinity. We concluded that the affinity towards the different peptides is mainly determined by the four amino acid motif CYFP present in the sequence of the template peptide. A higher affinity than that for the peptides is found for the parent proteins RBD and spike protein, which seems to be due to out of cavity effects caused by their larger footprint on the nanofilm surface.

Cytochrome c/polyelectrolyte multilayers investigated by E-QCM-D: effect of temperature on the assembly structure.

Protein multilayers, consisting of cytochrome c (cyt c) and poly(aniline sulfonic acid) (PASA), are investigated by electrochemical quartz crystal microbalance with dissipation monitoring (E-QCM-D). This technique reveals that a four-bilayer assembly has rather rigid properties. A thickness of 16.3 ± 0.8 nm is calculated with the Sauerbrey equation and is found to be in good agreement with a viscoelastic model. The electroactive amount of cyt c is estimated by the deposited mass under the assumption of 50% coupled water. Temperature-induced stabilization of the multilayer assembly has been investigated in the temperature range between 30 and 45 °C. The treatment results in a loss of material and a contraction of the film. The electroactive amount of cyt c also decreases during heating and remains constant after the cooling period. The contraction of the film is accompanied by the enhanced stability of the assembly. In addition, it is found that cyt c and PASA can be assembled at higher temperatures, resulting in the formation of multilayer systems with less dissipation.

Electrochemical Trimethylamine N-Oxide Biosensor with Enzyme-Based Oxygen-Scavenging Membrane for Long-Term Operation under Ambient Air.

An amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O2 reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O2 scavenging membrane. The TMAO sensor operates at a potential of -0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 µM and 15 mM, with a sensitivity of 2.75 ± 1.7 µA/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10% human serum, where the lowest detectable concentration is of 10 µM TMAO.

Redox properties and catalytic activity of surface-bound human sulfite oxidase studied by a combined surface enhanced resonance Raman spectroscopic and electrochemical approach.

Human sulfite oxidase (hSO) was immobilised on SAM-coated silver electrodes under preservation of the native heme pocket structure of the cytochrome b5 (Cyt b5) domain and the functionality of the enzyme. The redox properties and catalytic activity of the entire enzyme were studied by surface enhanced resonance Raman (SERR) spectroscopy and cyclic voltammetry (CV) and compared to the isolated heme domain when possible. It is shown that heterogeneous electron transfer and catalytic activity of hSO sensitively depend on the local environment of the enzyme. Increasing the ionic strength of the buffer solution leads to an increase of the heterogeneous electron transfer rate from 17 s(-1) to 440 s(-1) for hSO as determined by SERR spectroscopy. CV measurements demonstrate an increase of the apparent turnover rate for the immobilised hSO from 0.85 s(-1) in 100 mM buffer to 5.26 s(-1) in 750 mM buffer. We suggest that both effects originate from the increased mobility of the surface-bound enzyme with increasing ionic strength. In agreement with surface potential calculations we propose that at high ionic strength the enzyme is immobilised via the dimerisation domain to the SAM surface. The flexible loop region connecting the Moco and the Cyt b5 domain allows alternating contact with the Moco interaction site and the SAM surface, thereby promoting the sequential intramolecular and heterogeneous electron transfer from Moco via Cyt b5 to the electrode. At lower ionic strength, the contact time of the Cyt b5 domain with the SAM surface is longer, corresponding to a slower overall electron transfer process.

Amperometric biosensor based on coupling aminated laccase to functionalized carbon nanotubes for phenolics detection.

A biosensor for phenolic compounds based on a chemically modified laccase from Coriolus hirsuta immobilized on functionalized screen-printed carbon electrodes (SPCEs) was achieved. Different enzyme modifications and immobilization strategies were analyzed. The electrochemical response of the immobilized laccase on SPCEs modified with carboxyl functionalized multi-walled carbon nanotubes (COOH-MWCNT) was the highest when laccase was aminated prior to the adsorption onto the working electrode. The developed laccase biosensor sensitivity toward different phenolic compounds was assessed to determine the biosensor response with several phenolic compounds. The highest response was obtained for ABTS with a saturation value of Imax = 27.94 µA. The electrocatalytic efficiency (Imax/Kappm) was the highest for ABTS (5588 µA µM-1) followed by syringaldazine (3014 µA.µM-1). The sensors were considerably stable, whereby 99.5, 82 and 77% of the catalytic response using catechol as substrate was retained after 4, 8 and 10 successive cycles of reuse respectively, with response time average of 5 s for 12 cycles. No loss of activity was observed after 20 days of storage.

Electrocatalytic sulfite biosensor with human sulfite oxidase co-immobilized with cytochrome c in a polyelectrolyte-containing multilayer.

An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V (vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 microM sulfite with a sensitivity of 2.19 mA M(-1) sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.

Electrochemical MIP Sensor for Butyrylcholinesterase.

Molecularly imprinted polymers (MIPs) mimic the binding sites of antibodies by substituting the amino acid-scaffold of proteins by synthetic polymers. In this work, the first MIP for the recognition of the diagnostically relevant enzyme butyrylcholinesterase (BuChE) is presented. The MIP was prepared using electropolymerization of the functional monomer o-phenylenediamine and was deposited as a thin film on a glassy carbon electrode by oxidative potentiodynamic polymerization. Rebinding and removal of the template were detected by cyclic voltammetry using ferricyanide as a redox marker. Furthermore, the enzymatic activity of BuChE rebound to the MIP was measured via the anodic oxidation of thiocholine, the reaction product of butyrylthiocholine. The response was linear between 50 pM and 2 nM concentrations of BuChE with a detection limit of 14.7 pM. In addition to the high sensitivity for BuChE, the sensor responded towards pseudo-irreversible inhibitors in the lower mM range.

Electrocatalytically functional multilayer assembly of sulfite oxidase and cytochrome c.

An electrocatalytically functional multilayer has been designed using two proteins, cytochrome c and sulfite oxidase, and a polyelectrolyte (polyaniline sulfonate). The two proteins were co-immobilized on the surface of a gold electrode in alternating layers by electrostatic interactions using the layer-by-layer technique. The formation of this fully electro-active multilayer is characterized by quartz crystal microbalance and electrochemical experiments. The electro-catalytic characterization of the device containing up to 12 layers is based on generation of an oxidation current after sulfite addition. Besides the electron-transfer mechanism, the role of the different components in the electron-transport chain is clarified. Kinetic data were extracted to characterize the multilayer function. This artificial multilayer assembly is expected to be useful in the biosensor and biofuel cell development.