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This chapter explores the potential of vibration energy harvesting from structures leveraging flexoelectricity (i.e., strain gradient-induced polarization) and presents analytical modeling and analysis results for next-generation submicron scale energy harvesters. At such small geometric scales, flexoelectricity, as a gradient effect, enables significant electromechanical behavior even in nonpiezoelectric dielectric materials, while it enhances the electromechanical coupling in piezoelectric ones. In particular, energy harvesting is a potential future application area of flexoelectricity to enable ultra-low-power nanoscale devices by converting vibrations into electricity. The focus of this chapter is first placed on bending vibration of centrosymmetric cantilevers, such as a monolayer STO (Strontium Titanate) cantilever. An electroelastodynamic framework is presented and analyzed for flexoelectric power generation from strain gradient fluctuations in centrosymmetric dielectrics, by accounting for the presence of a finite electrical load across the surface electrodes as well as two-way electromechanical coupling, and capturing the size effect. Following recent efforts on the converse flexoelectric effect in finite samples, the proposed model properly accounts for a thermodynamically consistent, symmetric two-way coupling, i.e., the direct and converse effects. In addition to the electromechanical frequency response functions, the transverse mode flexoelectric coupling coefficient (a direct measure of energy conversion) is obtained analytically; its dependence on the cantilever thickness and a material figure of merit is shown. The modeling framework is then extended to noncentrosymmetric configurations, such as bimorph cantilevers made from Barium Titanate (BTO), to understand the interaction between flexoelectricity and piezoelectricity for different thickness levels from mm-scale to nm-scale, with a focus on resonant energy harvesting. The level of enhancement in the overall electromechanical coupling due to flexoelectricity with reduced thickness is quantified.

Summary The designed “ATCUN” motif (amino-terminal copper and nickel binding site) is a replica of naturally occurring ATCUN site found in many proteins/peptides, and an attractive platform for multiple applications, which include nucleases, proteases, spectroscopic probes, imaging, and small molecule activation. ATCUN motifs are engineered at periphery by conjugation to recombinant proteins, peptides, fluorophores, or recognition domains through chemically or genetically, fulfilling the needs of various biological relevance and a wide range of practical usages. This chemistry has witnessed significant growth over the last few decades and several interesting ATCUN derivatives have been described. The redox role of the ATCUN moieties is also an important aspect to be considered. The redox potential of designed M-ATCUN derivatives is modulated by judicious choice of amino acid (including stereochemistry, charge, and position) that ultimately leads to the catalytic efficiency. In this context, a wide range of M-ATCUN derivatives have been designed purposefully for various redox- and non-redox-based applications, including spectroscopic probes, target-based catalytic metallodrugs, inhibition of amyloid-β toxicity, and telomere shortening, enzyme inactivation, biomolecules stitching or modification, next-generation antibiotic, and small molecule activation., Graphical Abstract, Chemistry; Inorganic Chemistry; Biochemistry; Medical Biochemistry

In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b5 reductase was measured. Complex formation between both proteins suggests that cytochrome b5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death.

Cytochrome b5 reductase is an enzyme with the ability to generate superoxide anion at the expenses of NADH consumption. Although this activity can be stimulated by cytochrome c and could participate in the bioenergetic failure accounting in apoptosis, very little is known about other molecules that may uncouple the function of the cytochrome b5 reductase. Naphthoquinones are redox active molecules with the ability to interact with electron transfer chains. In this work, we made an inhibitor screening against recombinant human cytochrome b5 reductase based on naphthoquinone properties. We found that 5-hydroxy-1,4-naphthoquinone (known as juglone), a natural naphthoquinone extracted from walnut trees and used historically in traditional medicine with ambiguous health and toxic outcomes, had the ability to uncouple the electron transfer from the reductase to cytochrome b5 and ferricyanide. Upon complex formation with cytochrome b5 reductase, juglone is able to act as an electron acceptor leading to a NADH consumption stimulation and an increase of superoxide anion production by the reductase. Our results suggest that cytochrome b5 reductase could contribute to the measured energetic failure in the erythrocyte apoptosis induced by juglone, that is concomitant with the reactive oxygen species produced by cytochrome b5 reductase., This work was supported by the Associate Laboratory for Green Chemistry-LAQV which is financed by national funds from FCT/MCTES (UID/QUI/50006/2019). Experimental work was also supported by funding from Ayuda a Grupos de la Junta de Extremadura (Group GR18118) co-financed by the European Funds for Structural Development (FEDER). The authors also thank FCT for funds to GHTM (UID/Multi/04413/2013) and AKSA acknowledges FCT/MCTES for their “Investigador Doutorado” contracts' funding and signed with FCT/UNL in accordance with DL 57/2016 e Lei 57/2017.

NOTICE: this is the author’s version of a work that was accepted for publication in Electrochemistry Communications. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Electrochemistry Communications, VOL. 35, (October 2013). http://dx.doi.org/10.1016/j.elecom.2013.07.027 "This work reports the evaluation of a set of multi-walled carbon nanotubes (MWCNT) presenting different surface chemistries, as interfaces for the direct electrochemistry of the multihemic nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC27774 (Dd). The carbon nanotubes dispersions were prepared in aqueous media and deposited on pyrolytic graphite (PG) macroelectrodes, following a layer-by-layer methodology. The resulting MWCNT bed was coated with ccNiR and studied by cyclic voltammetry. Interestingly, although small non-catalytic cathodic waves were detected in all carbon nanotubes bioconjugates, the complexity of these electrochemical signals was partially deconvoluted in some materials, the less acidic ones emphasizing the contribution of the catalytic centre. Consistently, these MWCNT were the most favourable for enzyme catalysis, highlighting the importance of the surface oxide functionalities to enzyme reactivity."

A biosensor for nitrite analytical determination was developed using a cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 immobilized and electrically connected on a glassy carbon electrode by entrapment in an electrogenerated poly(pyrrole–viologen) matrix. The modified bioelectrode was studied by cyclic voltammetry and a catalytic current was observed in presence of nitrite. The linear range of the electrode response was 5.4–43.4 μM. The detection limit and the sensitivity were 5.4 μM and 1721 mA M−1 cm−2, respectively. The KMapp value determined from the Lineweaver–Burk plot was 86 μM. The biosensor fully maintained its electroenzymatic activity towards nitrite after four days. No catalytic response was observed in the presence of nitrate ions while interference from sulfites was considered negligible. Finally, the biosensor composition was optimized in term of monomer–enzyme ratio. Keywords: Biosensor, Polypyrrole, Nitrite reductase, Viologen, Enzyme wiring

Iron–sulfur clusters are ubiquitous and ancient prosthetic groups that are present in all kingdoms of life. They are flexible in terms of coordination and redox properties. The first function of these proteins to be identified was electron transfer back in the 1960s, but since then other functions have been discovered. Iron–sulfur centers are directly involved in catalysis, as sensors of gases (nitric oxide and oxygen), iron, and other cellular compounds, and also participate in biosynthetic pathways of iron–sulfur clusters from the basic to the more complex ones. The role of iron–sulfur clusters is emerging in DNA repair, as the driving force for DNA lesion recognition. In recent years, more and more enzymes containing iron–sulfur clusters have been identified, and it is now known that these centers catalyze a wide range of diverse reactions.

Mo- and W-enzymes are widely distributed in biology as they can be found in all domains of life. They perform key roles in several metabolic pathways catalyzing important reactions of the biogeochemical cycles of the more abundant elements of the earth. These reactions are usually redox processes involving the transfer of an atom from the substrate to the metal ion or vice versa. The Mo or W reactivity and specificity toward a substrate is determined by the polypeptide chain of the enzyme, which tunes the chemical properties of the metal ion. Two enzymes sharing almost identical active sites but catalyzing very different reactions are periplasmic nitrate reductase and formate dehydrogenase from bacteria. They represent a good example of how key changes in the amino acid sequence tune the properties of an enzyme. In order to analyze the chemistry of Mo and W in these enzymes, structural, kinetic and spectroscopic data are reviewed, along with the role of these enzymes in cell metabolism. In addition, the features that govern selectivity of metal uptake into the cell and Mo/W-cofactor biosynthesis are revised. Fil: González, Pablo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Quimica; Portugal Fil: Rivas, Maria Gabriela. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Quimica; Portugal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Mota, Cristiano S.. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Quimica; Portugal Fil: Brondino, Carlos Dante. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Moura, Isabel. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Quimica; Portugal Fil: Moura, José J.G.. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Quimica; Portugal

The redox behaviour of a ferredoxin (Fd) from Desulfovibrio alaskensis was characterized by electrochemistry. The protein was isolated and purified, and showed to be a tetramer containing one [3Fe–4S] and one [4Fe–4S] centre. This ferredoxin has high homology with FdI from Desulfovibrio vulgaris Miyazaki and Hildenborough and FdIII from Desulfovibrio africanus. From differential pulse voltammetry the following signals were identified: [3Fe-4S]+ 1/0 (E0′ = − 158 ± 5 mV); [4Fe–4S]+ 2/+1 (E0′ = − 474 ± 5 mV) and [3Fe–4S]0/− 2 (E0′ = − 660 ± 5 mV). The effect of pH on these signals showed that the reduced [3Fe–4S]0 cluster has a pKʹred′ = 5.1 ± 0.1, the [4Fe–4S]+ 2/+1 centre is pH independent, and the [3Fe–4S]0/−2 reduction is accompanied by the binding of two protons. The ability of the [3Fe–4S]0 cluster to be converted into a new [4Fe–4S] cluster was proven. The redox potential of the original [4Fe–4S] centre showed to be dependent on the formation of the new [4Fe-4S] centre, which results in a positive shift (ca. 70 mV) of the redox potential of the original centre. Being most [Fe–S] proteins involved in electron transport processes, the electrochemical characterization of their clusters is essential to understand their biological function. Complementary EPR studies were performed. Fil: Grazina, Raquel. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química. REQUIMTE/CQFB; Portugal Fil: Sousa, Patrícia M. Paes de. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química. REQUIMTE/CQFB; Portugal Fil: Brondino, Carlos Dante. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe; Argentina Fil: Carepo, Marta S. P.. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química. REQUIMTE/CQFB; Portugal Fil: Moura, Isabel. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química. REQUIMTE/CQFB; Portugal Fil: Moura, José J. G.. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química. REQUIMTE/CQFB; Portugal

ATP prevents G-actin cysteine oxidation and vanadyl formation specifically induced by decavanadate, suggesting that the oxometalate–protein interaction is affected by the nucleotide. The ATP exchange rate is increased by 2-fold due to the presence of decavanadate when compared with control actin (3.1 × 10 − 3 s − 1 ), and an apparent dissociation constant (k dapp ) of 227.4 ± 25.7 μM and 112.3 ± 8.7 μM was obtained in absence or presence of 20 μM V 10 , respectively. Moreover, concentrations as low as 50 μM of decameric vanadate species (V 10 ) increases the relative G-actin intrinsic fluorescence intensity by approximately 80% whereas for a 10-fold concentration of monomeric vanadate (V 1 ) no effects were observed. Upon decavanadate titration, it was observed a linear increase in G-actin hydrophobic surface (2.6-fold), while no changes were detected for V 1 (0–200 μM). Taken together, three major ideas arise: i) ATP prevents decavanadate-induced G-actin cysteine oxidation and vanadate reduction; ii) decavanadate promotes actin conformational changes resulting on its inactivation, iii) decavanadate has an effect on actin ATP binding site. Once it is demonstrated that actin is a new potential target for decavanadate, being the ATP binding site a suitable site for decavanadate binding, it is proposed that some of the biological effects of vanadate can be, at least in part, explained by decavanadate interactions with actin.

Aquatic Toxicology 63 (2003) 307-318 Metallothioneins (MT) were obtained after purification from metal-exposed clams (Ruditapes decussatus) using gelpermeation and ion-exchange chromatography. Four cadmium /metallothioneins (CdMTs) were resolved by ionexchange chromatography and they all had similar molecular weights, high cadmium content and an absorption spectra indicative of the presence of characteristic Cd /S aggregates. The NH2-terminal sequence suggests the presence of atleast two class I clam MT isoforms. For the other two putative clam CdMTs isolated, the results of the amino acid determination were inconclusive. One was slightly contaminated and the other one had a blocked NH2-terminal. These clam metalothioneins contain glycine, which seems to be a common feature of molluscan MT family and exhibited more similarity to oysters than to mussels. Further investigation on the inducibility of these isoforms will be necessary if clams are to be used as biomarkers of metal exposure.

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Publisher Summary This chapter focuses on simple and complex iron–sulfur-containing proteins isolated from sulfate reducing bacteria (SRB), to review the types and distribution of proteins, metal clusters involved and their association with other centers and cofactors, cluster binding motifs, electronic and magnetic properties of the iron-sulfur clusters present, and cluster interconversions, including heterometal cluster formation. The chapter describes the complex structures involving Fe–S basic units and other redox centers (flavin, heme, nickel, and molybdenum). Several three-dimensional crystal structures of simple and complex iron–sulfur-containing proteins isolated from sulfate reducers have been solved. The main structural features have been discussed in the chapter. They include the simple rubredoxin family ferredoxins, hydrogenase, and AOR. In addition, the structures of other electron carriers, such as cytochromes and flavodoxins, have been determined. The overall data indicate the wide range of structural solutions adopted in the construction of the metal-containing active sites and were inspiring motifs for the proposal of catalytic mechanisms.

Publisher Summary This chapter reviews adenylylsulfate (APS) reductases from sulfate-reducing bacteria (SRB). The sulfate molecule is inert chemically and must be activated in order to enter any of the pathways. All the organisms that utilize sulfate contain the enzyme ATP-sulfurylase, which catalyzes the formation of adenylylsulfate and inorganic pyrophosphate (PP i ) from ATP and sulfate. The APS molecule has twice the energy of the comparable ADP molecule, and the equilibrium of the reaction lies in the direction of ATP and sulfate. An inorganic pyrophosphatase shifts the equilibrium toward APS formation by hydrolyzing the PP i molecules. The key reaction in assimilatory sulfate reduction that differentiates it from dissimilatory sulfate reduction is the transferring of the sulfonate group of APS or 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to a thiol group producing the corresponding nucleotide (AMP or PAP) plus a thiosulfonate. This thiol is either a low molecular weight compound such as glutathione or a protein such as thioredoxin or glutaredoxin. The purification of APS reductase must be carried out in such a way that it maximizes its final purity and yet minimizes the time required to accomplish it.

Publisher Summary This chapter describes aldehyde oxidoreductases and other molybdenum (Mo)-containing enzymes. Desulfovibrio gigas ( D. gigas ) Mo -containing protein is isolated on the basis of its optical absorption characteristics.Mo was first detected by electron paramagnetic resonance (EPR). All purification operations for aldehyde oxidoreductases are carried out at 4, starting from a D. gigas NCIB 9332 cell-free crude extract. Activity is determined aerobically at 25 by measuring the rate of 2, 6-dichlorophenol-indophenol (DCPIP) reduction at 600 nm ) in a 1-cm optical path spectrophotometer quartz cell. A pH profile of the enzyme activity is determined in different buffer systems, such as citrate, phosphate, Tris-HCl, and Tris-glycine—covering a pH range of 4–11. DCPIP and ferricyanide are shown to be capable of cycling the electronic flow, whereas other cation and anion dyes do not. The visible absorption spectrum of the protein is similar to that observed for the deflavo forms of xanthine and aldehyde oxidases and reminiscent of the one observed for plant-type ferredoxins.