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Background: Facial filling based on hyaluronic acid (HA) is an esthetic procedure that is widely performed worldwide. Methodology: Fifty patients underwent facial filling based on HA, and 10 boardcertified dermatologists evaluated clinical photographs before and after the procedure and completed a questionnaire to provide information about the procedure. Results: All doctors and 96% of patients were agreed about the pre- and postprocedural photographs. Physicians were more aware of the possibility of frequent pain and erythema. Both groups showed agreement in their responses on the following topics: the possibility of hematoma and edema, a lack of secretion, improvement in patients' self- confidence, recognition of the procedure by third parties, and knowledge of the procedure by the dermatologists themselves. However, physicians tend to overestimate the possibility of erythema, pain, and edema; they also tend to minimize the duration and discomfort of postprocedural edema and the risk of third parties noticing patients' HA filling and how much it bothers patients when third parties do notice. Conclusion: Doctors and patients disagree on some questions about what actually happens and what is supposed to happen in facial filling procedures based on HA. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Fluorescence emission of a fluorescein derivative decreased in acid pH. • Fluorescence anisotropy of fluorescein derivative increased in acid pH. • The monoanionic species of fluorescein derivative predominates under physiological pH. • A constant fluorescence emission of fluorescein derivative is a prerequisite for binding studies. Abstract Fluorescence spectroscopy is a powerful tool to comprehend macromolecules interaction. The characteristics of the fluorophore as well its behavior under the experimental conditions are central to ascertain the results. The present work characterizes the fluorescence emission and anisotropy of a fluorescein derivative linked to a double strand DNA containing 67 base pairs (named as 6-FAM-67-mer) under different pH conditions. The results show that fluorescence emission and anisotropy of this fluorescein derivative varies according to pH (as the pH becomes more acidic, the total fluorescence emission decreases). The decrease in fluorescence emission intensity is accompanied by an increase in anisotropy. The monoanionic form of fluorescein derivative is the predominant species from pH 7.5 to 9.2. Our main conclusion is that a constant fluorescein derivative emission is a prerequisite to determine association constants between macromolecules. [ABSTRACT FROM AUTHOR]

Living organisms use selenium mainly in the form of selenocysteine in the active site of oxidoreductases. Here, selenium's unique chemistry is believed to modulate the reaction mechanism and enhance the catalytic efficiency of specific enzymes in ways not achievable with a sulfur-containing cysteine. However, despite the fact that selenium/sulfur have different physicochemical properties, several selenoproteins have fully functional cysteine-containing homologues and some organisms do not use selenocysteine at all. In this review, selected selenocysteine-containing proteins will be discussed to showcase both situations: (i) selenium as an obligatory element for the protein's physiological function, and (ii) selenium presenting no clear advantage over sulfur (functional proteins with either selenium or sulfur). Selenium's physiological roles in antioxidant defence (to maintain cellular redox status/hinder oxidative stress), hormone metabolism, DNA synthesis, and repair (maintain genetic stability) will be also highlighted, as well as selenium's role in human health. Formate dehydrogenases, hydrogenases, glutathione peroxidases, thioredoxin reductases, and iodothyronine deiodinases will be herein featured. [ABSTRACT FROM AUTHOR]

Purpose: To identify cardiovascular risk factors in women between 40 and 65 years old in two private clinic of gynecology in Minas Gerais. Methods: Cross-sectional study from January/2016 to January/2017 in 137 climacteric women. A standardized questionnaire was used to evaluate socio-demographic characteristics, climacteric phases, cardiovascular risk factors, menopause (age, time and type), number of pregnancies, normal births, abortions, partners and hormonal therapy. The evaluation model was used according to the Framingham risk score. Results: The median age was 47 years, and 35% were postmenopausal. The majority were married and 42.3% caucasian. Abdominal circumference greater than 80 cm was observed in 87.6%, and greater than 88 cm in 67.2%. High total cholesterol (TC) was observed in 48.2%. Low levels of high-density lipoprotein (HDL-c) were observed in 42.3%. Elevated levels were observed for low density lipoprotein (LDL-c) in 39.4%, triglycerides in 29.9%, and fasting glucose in 8.8%. Also, systolic and diastolic blood pressure were considered high in 25.5% cases. Postmenopausal women presented higher values of TC and HDL-c. Analysis of the Framingham risk score revealed a higher cardiovascular risk for postmenopausal women. Higher values of waist circumference, glycemia and triglycerides were observed for women with higher cardiovascular risk. Conclusions: Postmenopausal women had a higher risk of cardiovascular events when compared to premenopausal ones. [ABSTRACT FROM AUTHOR]

The entrapment of quantum dots (QDs) in the inner part of micelles formed by surfactant polymers is a powerful methodology to prepare stable and photoluminescent core nanoparticles with enhanced optical properties. These features are crucial for the application of QDs in the design of hybrid assemblies for light harvesting applications, where energy transfer processes are required. The present work was focused on the synthesis of a surfactant homopolymer, poly (acrylic acid) (PAA) macroRAFT, to be used as a stabilizer of hydrophobic cadmium telluride (CdTe) QDs in aqueous solution. PAA macroRAFT was synthesized by reversible addition-fragmentation chain-transfer (RAFT) polymerization in a single chemical reaction. Its micelles were used to entangle and entrap hydrophobic CdTe QDs, with different molar ratio of polymer and QDs. The morphology and optical properties of the entrapped QDs were determined. The results showed that PAA macroRAFT is able to form micelles with a critical micelle concentration of 2.08 mg/mL. It was also noticed that the molar ratio of polymer and QDs have high influence on the QDs' morphology and their optical properties. The QDs' photoluminescence quantum yield was enhanced approximately 23% upon their entrapment in PAA macroRAFT micelles, using 60 equivalents of polymer. Moreover, while in solution, QDs are well-dispersed, having a 3.5 nm diameter, upon being entrapped in the micelles, tend to form clusters with a size around 100 nm. [ABSTRACT FROM AUTHOR]

The global energy demand and the present high dependence on fossil fuels have caused an unprecedented increase in the Earth’s atmosphere carbon dioxide concentration. Its exponential and uncontrollable rise is responsible for large and unpredictable impacts on the world climate and for ocean acidification, thus, being a major concern for the ecosystems and human’s daily life. On the other hand, the carbon dioxide abundance and low cost make it an interesting source for the production of chemical feedstocks and fuels. Yet, the thermodynamic and kinetic stability of the carbon dioxide molecule makes its utilization a laboratorial/industrially challenging task. In this Review, we propose to use the molybdenum and tungsten-containing formate dehydrogenase (FDH) enzymes as a model to understand the mechanistic strategies and key chemical features needed to reduce carbon dioxide to formate. We will highlight the present knowledge about the structure of FDHs, with particular emphasis on active site features, reaction mechanism and ability to reduce carbon dioxide to formate. The information gathered aims to inspire the development of new efficient (bio)catalysts for the atmospheric carbon dioxide utilization, to produce energy and chemical feedstocks, while reducing an important environmental pollutant. [ABSTRACT FROM AUTHOR]

A linear cluster formulated as [S 2 MoS 2 CuS 2 MoS 2 ] 3 − , a unique heterometallic cluster found in biological systems, was identified in a small monomeric protein (named as Orange Protein). The gene coding for this protein is part of an operon mainly present in strict anaerobic bacteria, which is composed (in its core) by genes coding for the Orange Protein and two ATPase proposed to contain Fe-S clusters. In Desulfovibrio desulfuricans G20, there is an ORF, Dde _ 3197 that encodes a small protein containing several cysteine residues in its primary sequence. The heterologously produced Dde_3197 aggregates mostly in inclusion bodies and was isolated by unfolding with a chaotropic agent and refolding by dialysis. The refolded protein contained sub-stoichiometric amounts of iron atoms/protein (0.5 ± 0.2), but after reconstitution with iron and sulfide, high iron load contents were detected (1.8 ± 0.1 or 3.4 ± 0.2) using 2- and 4-fold iron excess. The visible absorption spectral features of the iron-sulfur clusters in refolded and reconstituted Dde_3197 are similar and resemble the ones of [2Fe-2S] cluster containing proteins. The refolded and reconstituted [2Fe-2S] Dde_3197 are EPR silent, but after reduction with dithionite, a rhombic signal is observed with g max = 2.00, g med = 1.95 and g min = 1.92, consistent with a one-electron reduction of a [2Fe-2S] 2 + cluster into a [2Fe-2S] 1 + state, with an electron spin of S = ½. The data suggests that Dde_3197 can harbor one or two [2Fe-2S] clusters, one being stable and the other labile, with quite identical spectroscopic properties, but stable to oxygen. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) has a significant impact on sickle cell disease (SCD) morbidity and mortality. Early identification of individuals at highest risk of developing CKD may allow therapeutic intervention to prevent worse outcomes. This study aimed to evaluate the prevalence and risk factors for reduced estimated glomerular filtration rate (eGFR) among adults with SCD in Brazil. Participants in the REDS-III multicenter SCD cohort with more severe genotypes aged ≥ 18 years with at least two serum creatinine values were analyzed. The eGFR was calculated using the Jamaica Sickle Cell Cohort Study GFR equation. The eGFR categories were defined according to the K/DOQI. Participants with eGFR ≥ 90 were compared to those with those with eGFR < 90. Among the 870 participants, 647 (74.4%) had eGFR ≥ 90, 211 (24.3%) had eGFR 60 to 89, six (0.7%) had eGFR 30 to 59, and six (0.7%) had ESRD. Male sex (OR: 37.3; 95%CI: 22.4–65.1), higher age (OR: 1.04; 95%CI: 1.02–1.06), higher diastolic blood pressure (OR: 1.03; 95%CI: 1.009–1.06), lower Hb (OR: 0.80; 95%CI: 0.68–0.93), and lower reticulocytes (OR: 0.94; 95%CI: 0.89–0.99) levels were independently associated with eGFR < 90. There was a trend towards higher odds of death in participants with eGFR < 90 (OR: 1.8; 95%CI: 0.95–3.32; p = 0.065). In turn, participants with eGFR < 60 had a 12.2 (95%CI: 2.1–96.9) times higher odds for death when compared to those with eGFR ≥ 60. In this study, eGFR < 90 was observed in one-quarter of adults. Older age, male sex, higher diastolic blood pressure, lower hemoglobin, and lower reticulocyte levels were associated with occurrence of eGFR < 90. Estimated GFR < 60 increased the risk of mortality. [ABSTRACT FROM AUTHOR]

Identifying redox partners and the interaction surfaces is crucial for fully understanding electron flow in a respiratory chain. In this study, we focused on the interaction of nitrous oxide reductase (NOR), which catalyzes the final step in bacterial denitrification, with its physiological electron donor, either a c-type cytochrome or a type 1 copper protein. The comparison between the interaction of NOR from three different microorganisms, Pseudomonas nautica, Paracoccus denitrificans, and Achromobacter cycloclastes, with their physiological electron donors was performed through the analysis of the primary sequence alignment, electrostatic surface, and molecular docking simulations, using the bimolecular complex generation with global evaluation and ranking algorithm. The docking results were analyzed taking into account the experimental data, since the interaction is suggested to have either a hydrophobic nature, in the case of P. nautica NOR, or an electrostatic nature, in the case of P. denitrificans NOR and A. cycloclastes NOR . A set of well-conserved residues on the NOR surface were identified as being part of the electron transfer pathway from the redox partner to NOR (Ala495, Asp519, Val524, His566 and Leu568 numbered according to the P. nautica NOR sequence). Moreover, we built a model for Wolinella succinogenes NOR, an enzyme that has an additional c-type-heme-containing domain. The structures of the NOR domain and the c-type-heme-containing domain were modeled and the full-length structure was obtained by molecular docking simulation of these two domains. The orientation of the c-type-heme-containing domain relative to the NOR domain is similar to that found in the other electron transfer complexes. [ABSTRACT FROM AUTHOR]

Computer Science (CS) introductory courses that are offered by the Department of Information Systems at the University of Minho (UM), Portugal, seem to abound in non-motivated students. They are characterized by high failure and withdrawal rates and use mainly deductive teaching approaches. Deductive instruction begins with theories and progresses to applications of those theories. Active approaches to teaching are more inductive. Inductive instruction begins with the presentation of, for instance, a specific problem that introduces a topic, and theories are studied on a need-to-know basis. This pilot study describes how active learning techniques have been successfully applied to a CS introductory course, reducing its failure and withdrawal rates. The study portrays the changes in the course from teacher-centered education to a learner-centered approach, using two different editions of the same course, the first one being teacher-centered and the second learner-centered. The results in terms of success, failure, and dropout are given and the impact of a more student-centered approach on student involvement in learning is analyzed. The authors discuss implications of student-centered learning for the classroom and pay attention to some of the drawbacks of an implementation more focused on active learning. [Copyright &y& Elsevier]

Abstract: Direct electrochemical response was first time observed for the redox centers of Desulfovibrio gigas [NiFe]-Hase, in non-turnover conditions, by cyclic voltammetry, in solution at glassy carbon electrode. The activation of the enzyme was achieved by reduction with H2 and by electrochemical control and electrocatalytic activity was observed. The inactivation of the [NiFe]-Hase was also attained through potential control. All electrochemical data was obtained in the absence of enzyme inhibitors. The results are discussed in the context of the proposed mechanism currently accepted for activation/inactivation of [NiFe]-Hases. [Copyright &y& Elsevier]

Metalloenzymes control enzymatic activity by changing the characteristics of the metal centers where catalysis takes place. The conversion between inactive and active states can be tuned by altering the coordination number of the metal site, and in some cases by an associated conformational change. These processes will be illustrated using heme proteins (cytochrome c nitrite reductase, cytochrome c peroxidase and cytochrome cd 1 nitrite reductase), non-heme proteins (superoxide reductase and [NiFe]-hydrogenase), and copper proteins (nitrite and nitrous oxide reductases) as examples. These examples catalyze electron transfer reactions that include atom transfer, abstraction and insertion. [ABSTRACT FROM AUTHOR]

A novel iron–sulfur containing protein, a ferredoxin (Fd), was purified to homogeneity from the extract of Desulfovibrio desulfuricans American type culture collection (ATCC) 27774. The purified protein is a 13.4 kDa homodimer with a polypeptide chain of 60 amino acids residues, containing eight cysteines that coordinate two [4Fe–4S] clusters. The protein is shown to be air sensitive and cluster conversions take place. We structurally characterize a redox state that contains two [4Fe–4S] cores. 1D and 2D 1H NMR studies are reported on form containing the clusters in the oxidized state. Based on the nuclear Overhauser effect (NOE), relaxation measurements and comparison of the present data with the available spectra of the analogous 8Fe Fds, the cluster ligands were specifically assigned to the eight-cysteinyl residues. [Copyright &y& Elsevier]

Examines the nature and properties of the complex electron-transfer chain formed between flavodoxin and cytochrome c3. Nuclear magnetic resonance protein titrations; Prediction of protein structure by homology modeling; Analysis of the surface electrostatic potentials; Computer graphics modeling of the complex.

Methanosarcina barkeri ferredoxin was purified and characterized by electron paramagnetic resonance (EPR) and Mössbauer spectroscopy. The purification procedure included chromatographic steps on IDEAE-cellulose and gel filtration. The isolated protein is unstable under aerobic conditions. The ferredoxin exhibits charge transfer bands at 283 nm and 405 nm with an absorption ratio A405/A233 = 0.73. Its molecular weight has been estimated to be 20000-22000 by gel filtration chromatography. The native ferredoxin exhibits an intense EPR signal at g = 2.02 and only a very weak g = 1.94 signal develops upon reduction with dithionite. The Mössbauer spectra of the reduced protein are characteristic of a [3 Fe-3 S] center. The combined EPR and Mössbauer studies show that M. barkeri ferredoxin contains only [3 Fe-3S] clusters, similar to Azotobacter vinelandii Fd[Emptage, M. H., Kent,T. A., Huynh, B. H., Rawlings,J., Orme-Johnson. W. H. & Münck, M. (1980) J. Biol. Chem. 255. 1793-1796], Desulfovibrio gigas FdII [Huynh, B. H., Moura, J. J. G., Moura, I., Kent, T. A., LeGall, J., Xavier, A. V. & Münck, E. (1980) J. Blot Chem. 255, 3242-3244] and mitochondrial beef heart aconitase [Kent, T. A., Dreyer, J.-L., Kennedy, M. C., Huynh, B. H., Emptage, M. H., Beinert, H. & Münck, E. (1982) Proc. Nail Acad. Sci. USA, 79, 1096-1100]. [ABSTRACT FROM AUTHOR]

Mössbauer spectroscopy was used to study the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Samples with different degrees of reduction were prepared using a redox-titration technique. In the reduced cytochrome c3, all four hemes are reduced and exhibit diamagnetic Mössbauer spectra typical for low-spin ferrous hemes (S = 0). In the oxidized protein, the hemes are low-spin ferric (S = ½) and exhibit overlapping magnetic Mössbauer spectra. A method of differential spectroscopy was applied to deconvolute the four overlapping heme spectra and a crystal-field model was used for data analysis. Characteristic Mössbauer spectral components for each heme group are obtained. Hyperfine and crystal-field parameters for all four hemes are determined from these deconvoluted spectra. [ABSTRACT FROM AUTHOR]

The soluble (cytoplasmic plus periplasmic) Ni/Fe-S/Se-containing hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from cells grown in an 57Fe-enriched medium, and its iron-sulfur centers were extensively characterized by Mössbauer and EPR spectroscopies. The data analysis excludes the presence of a [3Fe-4S] center, either in the native (as isolated) or in the hydrogen-reduced states. In the native state, the non-heine iron atoms arc arranged as two diamagnetic [4Fe-4S]2+ centers. Upon reduction, these two centers exhibit distinct and unusual Mössbauer spectroscopic parameters. The centers were found to have similar mid-point potentials (≈ - 315 mV) as determined by oxidation-reduction titrations followed by EPR. [ABSTRACT FROM AUTHOR]

In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (≈ 400 kDa). It is an iron-sulfur, ravincontaining protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10-0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin—spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and II). Mössbauer studies on 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential ≤ 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical Mössbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin-type [4Fe-4S] cluster, suggesting a different structure and/or a distinct cluster-ligand environment. [ABSTRACT FROM AUTHOR]

When purified, a high-potential c-type monohaem cytochrome from the nitrate-respiring organism, Wollinella succinogenes (VPI 10659), displayed a minimum molecular mass of 8.2 kDa and 0.9 mol iron and 0.95 mol haem groups/mol protein. Visible light spectroscopy suggested the presence of an equilibrium between two ligand arrangements around the haem, i.e. an absorption band at 695 nm characteristic of haem-methionine coordination (low-spin form) coexisting with a high-spin form revealed by a band at 619 nm and a shoulder at 498 nm. The mid-point redox potential measured by visible redox titration of the low-spin form was approximately +100 mV. Binding cyanide (Ka = 5×105 M¹) resulted in the displacement of the methionyl axial residue, and full conversion to a low-spin, cyanide-bound form. Structural features were studied by 300-MHz ¹H-NMR spectroscopy. In the oxidized state, the pH dependence of the haem methyl resonances (pH range 5–10) and the magnetic susceptibility measurements (using an NMR method) were consistent with the visible light spectroscopic data for the presence of a high-spin/low-spin equilibrium with a transition pKa of 7.3. The spin equilibrium was fast on the NMR time scale. The haem methyl resonances presented large downfield chemical shifts. An unusually broad methyl resonance at around 35 ppm (pH = 7.5, 25°C) was extremely temperature-dependent [δ(323 K)— δ(273K) = 7.2 ppm] and was assigned to the S-CH3 group of the axial methionine. In the ferrous state only a low-spin form is present. The haem meso protons, the methyl group and the methylene protons from the axial methionine were identified in the reduced form. The resonances from the aromatic residues (three tyrosines and one phenylalanine) were also assigned. Detailed monitoring of the NMR-redox pattern of the monohaem cytochrome from the fully reduced up to the fully oxidized state revealed that the rate of the intermolecular electronic exchange process was approximately 6×106 M-1 s-1 at 303 K and pH = 6.31. A dihaem cytochrome also present in the crude cell extract and purified to a homogeneous state, exhibited a molecular mass of 11 kDa and contained 2.43 mol iron and 1.89 mol haem c moieties/mol cytochrome. The absorption spectrum in the visible region exhibited no band at 695 nm, suggesting that methione is not a ligand for either of the two haems. Recovery of only small amounts of this protein prevented more detailed structural analyzes. [ABSTRACT FROM AUTHOR]

An EPR redox titration was performed on the tetraheme cytochrome cs isolated from Desulfovibrio baculatus (strain 9974), a sulfate-reducer. Using spectral differences at different poised redox states of the protein, it was possible to individualize the EPR g-values of each of the four hemes and also to determine the mid-point redox potentials of each individual heme: heme 4 (-70 mV) at gmax = 2.93, gmed = 2.26 and gmin = 1.51; heme 3 (-280 mV) at gmax = 3.41; heme 2 (-300 mV) at gmax = 3.05, gmed = 2.24 and gmin = 1.34; and heme 1 (-355 mV) at gmax = 3.18. A previously described multi-redox equilibria model used for the interpretation of NMR data of D. gigas cytochrome c3 [Santos, H., Moura, J. J. G., Mourn, I., LeGall, J. & Xavier, A. V. (1984) Eur. J. Biochem. 141, 283-296] is discussed in terms of the EPR results. [ABSTRACT FROM AUTHOR]

Two c-type cytochromes were purified and characterized by electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopic techniques, from the sulfate-reducer nitrogen-fixing organism, Desulfovibrio desulfuricans strain Berre-Eau (NCIB 8387). The purification procedures included several chromatographic steps on alumina, carboxymethylcellulose and gel filtration. A tetrahaem and a monohaem cytochrome were identified. The multihaem cytochrome has visible, EPR and NMR spectra with general properties similar to other low-potential bis-histidinyl axially bound haem proteins, belonging to the class of tetrahaem cytochrome c3 isolated from other Desulfovibrio species. The monohaem cytochrome c553 is ascorbate-reducible and its EPR and NMR data are characteristic of a cytochrome with methionine-histidine ligation. Their properties are compared with other homologous proteins isolated from sulfate-reducing bacteria. [ABSTRACT FROM AUTHOR]

A dihaem cytochrome (Mr 37400) with cytochrome c peroxidase activity was purified from Pseudomonas stutzeri (ATCC 11607). The haem redox potentials are far apart: one of the haems is completely ascorbate-reducible and the other is only reduced by dithionite. The coordination, spin states and redox properties of the covalently bound haems were probed by visible, NMR and electron paramagnetic resonance (EPR) spectroscopies in three oxidation states. In the oxidized state, the low-temperature EPR spectrum of the native enzyme is a complex superimposition of three components: (I) a low-spin haem indicating a histidinyl-methionyl coordination; (II) a low-spin haem indicating a histidinyl-histidinyl coordination; and (III) a minor high-spin haem component. At room temperature, NMR and optical studies indicate the presence of high-spin and low-spin haems, suggesting that for one of the haems a high-spin to low-spin transition is observed when temperature is decreased. In the half-reduced state, the component I (high redox potential) of the EPR spectrum disappears a nd induces a change in the g-values and linewidth of component II; the high-spin component II is no longer detected at low temperature. Visible and NMR studies reveal the presence of a high-spin ferric and a low-spin (methionylcoordinated) ferrous state. The NMR data fully support the haem-haem interaction probed by EPR. In the reduced state, the NMR spectrum indicates that the low-potential haem is high-spin ferrous. [ABSTRACT FROM AUTHOR]

Pseudomonas perfectomarinus (ATCC 14405) dihaem cytochrome c552 was studied by 300-MHz proton magnetic resonance. Some of the haem resonances were assigned in the fully reduced and fully oxidized states. No evidence was found for methionine haem axial coordination. The oxidation-reduction equilibrium was studied in detail. Due to the large difference in mid-point redox potential between the two haems (+174 mV, for haem II and -180 mV for haem I) an intermediate oxidation state could be obtained containing reduced haem I and oxidized haem II. In this way the total paramagnetic shift at different oxidation levels could be decomposed in the intrinsic and extrinsic contributions. It was found that the two haems interact. The rate of electron exchange is slow on the NMR time scale. The redox equilibria are discussed for four possible redox species in solution. [ABSTRACT FROM AUTHOR]

Below 30 K, oxidized Desulfovibrio gigas hydrogenase presents an intense electron paramagnetic resonance (EPR) signal centered at g = 2.02, typical of an iron-sulfur center. In addition a rhombic EPR signal, attributed to Ni(III) species, is also observed [LeGall, J., Ljungdahl, P., Moura, I., Peck, H. D., Jr, Xavier, A. V., Moura, J. J. G., Teixeira, M., Huynh, B. H., and DerVartanian, D. V. (1982) Biochem. Biophys. Res. Commun. 106, 610-616; and Cammack, R., Patil, D., Aguirre, R., and Hatchikian, E. C., (1982) FEBS Lett. 142, 289-292]. At higher temperatures (77 K) the iron-sulfur EPR signal is broader and all the EPR features of the rhombic nickel signal can easily be observed. We have now obtained additional information concerning the redox properties of these EPR active centers, using an EPR redox titration method in the presence of dye mediators at pH = 8.5. The mid-point potential was determined to be -70 mV for the Fe,S cluster and -220 mV for the Ni center. Intermediate oxidation states were obtained upon partial reduction with either dithionite or hydrogen. Although upon dithionite reduction the centers are reduced in the order of decreasing mid-point reduction potentials, under a hydrogen atmosphere the nickel center reduces preferentially. This suggests a catalytic involvement of the nickel redox center in the binding of hydrogen. Preliminary Mössbauer studies on Desulfovibrio gigas hydrogenase reveal the presence of a paramagnetic 3 Fe center and two 4 Fe centers. The 3 Fe center is responsible for the g = 2.02 EPR signal but the two 4 Fe centers have been so far undetectable by EPR. [ABSTRACT FROM AUTHOR]

Keywords: Anti-aging; chemical peel; collagen; cosmetic dermatology; neocollagenesis EN Anti-aging chemical peel collagen cosmetic dermatology neocollagenesis 1766 1769 4 04/06/22 20220401 NES 220401 Dear editor, Phenol-croton oil peel generates skin neocollagenesis. D0 or A, Immediately before the procedures; D90±2 or B, 90±2 days after the procedure; MMP-1, Metalloproteinase type-1; MMP-2, Metalloproteinase type-2; JunB, Transcriptor factor JunB; p53, Protein p53; C, Immunohistochemistry pictures. Neocolagenesis by phenol-croton oil peel is independent from the MMP - 1/MMP-2/JunB and p53 pathways. [Extracted from the article]

Copper-containing nitrous oxide reductase catalyzes a 2-electron reduction of the green-house gas N2O to yield N2. It contains two metal centers, the binuclear electron transfer site CuA, and the unique, tetranuclear CuZ center that is the site of substrate binding. Different forms of the enzyme were described previously, representing variations in oxidation state and composition of the metal sites. Hypothesizing that many reported discrepancies in the structural data may be due to radiation damage during data collection, we determined the structure of anoxically isolated Marinobacter nauticus N2OR from diffraction data obtained with low-intensity X-rays from an in-house rotating anode generator and an image plate detector. The data set was of exceptional quality and yielded a structure at 1.5 Å resolution in a new crystal form. The CuA site of the enzyme shows two distinct conformations with potential relevance for intramolecular electron transfer, and the CuZ cluster is present in a [4Cu:2S] configuration. In addition, the structure contains three additional types of ions, and an analysis of anomalous scattering contributions confirms them to be Ca2+, K+, and Cl–. The uniformity of the present structure supports the hypothesis that many earlier analyses showed inhomogeneities due to radiation effects. Adding to the earlier description of the same enzyme with a [4Cu:S] CuZ site, a mechanistic model is presented, with a structurally flexible CuZ center that does not require the complete dissociation of a sulfide prior to N2O binding. The [4Cu:2S] CuZ site in M. nauticus N 2O reductase. The electron density map shown is contoured at the 5 σ level, highlighting the presence of two sulfide ligands. 705x677mm (72 x 72 DPI) [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • N 2 O global warming impact is 300× higher than CO 2. • Nitrous oxide reductase has two copper centers, CuA and "CuZ". • "CuZ" center can be isolated as CuZ*(4Cu1S) or as CuZ(4Cu2S). • CuZ*(4Cu1S) [4Cu1+] turnover number explains high reduction rate of whole-cells. • Structural and functional model compounds for nitrous oxide reductase active site. Abstract Nitrous oxide is a potent greenhouse gas with a global warming impact 300-fold higher than carbon dioxide. Due to its exponential increase in the atmosphere and its implications in climate change there is the need to develop strategies to mitigate its emissions and to reduce it to the inert dinitrogen gas. Only three enzymes have been reported to be able to reduce nitrous oxide, namely nitrogenase, one multicopper oxidase and nitrous oxide reductase, with the latter being the only one with a relevant physiological activity. In this enzyme, reduction of nitrous oxide occurs in a unique catalytic tetranuclear sulfide center, named "CuZ" center, a complex center required to overcome the high activation barrier of this reaction. Nitrous oxide reductase can be isolated with "CuZ" center in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S), that differ in their catalytic and spectroscopic properties. Recently, another step towards a better understanding of the catalytic and activation mechanism of this enzyme was taken by identifying and spectroscopically characterizing an intermediate species of its catalytic cycle, CuZ0. A different approach for N 2 O reduction can be attained using model compounds. The unique structural motif present in "CuZ" center, a Cu 4 (µ 4 -S), has been a challenge for inorganic synthesis but several synthetic clusters that mimic different forms of "CuZ" center have been reported. Model compounds for the oxidation states involved in N 2 O reduction are also available. The advances in this area will be discussed in light of the recent data, with structural and functional model compounds of N 2 OR active site. [ABSTRACT FROM AUTHOR]

The nitration of tyrosine residues in proteins represents a specific footprint of the formation of reactive nitrogen species (RNS) in vivo. Here, the fusion product of orange protein (ATCUN‐ORP) was used as an in vitro model system containing an amino terminal Cu(II)‐ and Ni(II)‐binding motif (ATCUN) tag at the N‐terminus and a native tyrosine residue in the metal‐cofactor‐binding region for the formation of 3‐NO2‐Tyr (3‐NT). It is shown that NiII‐ATCUN unusually performs nitration of tyrosine at physiological pH in the presence of the NO2−/SO32−/O2 system, which is revealed by a characteristic absorbance band at 430 nm in basic medium and 350 nm in acidic medium (fingerprint of 3‐NT). Kinetics studies showed that the formation of 3‐NT depends on sulfite concentration over nitrite concentration suggesting key intermediate products, identified as oxysulfur radicals, which are detected by spin‐trap EPR study by using 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO). This study describes a new route in the formation of 3‐NT, which is proposed to be linked with the sulfur metabolism pathway associated with the progression of disease occurrence in vivo. NiII‐ATCUN in action! The NiII ions bound to the amino terminal Cu(II)‐ and Ni(II)‐binding (ATCUN) motifs in orange protein unusually perform nitration of tyrosine at physiological pH in the presence of the NO2−/SO32−/O2 system over the H2O2/NO2− system. This result may provide the sulfur metabolism pathway to yield 3‐nitrotyrosine, which may be associated with sulfur‐related human diseases in vivo. [ABSTRACT FROM AUTHOR]

Rubredoxins (Rds) and their derivatives have been extensively used, in the last few decades, in order to elucidate structure and functional aspects of metal sites involving rich sulphur coordination spheres. The derivatives have been designed with two main purposes: (a) replacement of selective and specific amino acid residues in native systems by site direct mutagenesis, and (b) replacement of native metal ion (iron), producing novel metal sites. We will highlight in this review the key amino acid residues, recognized in native Rds, directly involved in electron transfer mechanisms. By protein template assisted synthesis, metal-substituted Rds are used as structural probes and bio-models. The tetra-cysteinyl metal coordination site in Rd has the surprising capacity of chelating a wide variety of metal ions (other than native iron), and this chemistry has been witnessing significant growth based on two main interests: (i) metals such as 57 Fe, Zn, Co, Cd, Ga, In, and Hg were introduced and derivatives were synthesized in order to specifically probe structural determinants imposed by the protein, and (ii) Zn, Ni, Cu, as well as Mo (and most recently, W), were used with a particular interest: to model complex metal centres in sulphur-rich enzymes. The latter, in particular, is discussed in the context of Rd serving as a model complex of native enzymes, as well as the synthesis of small inorganic complexes. The review aims to bring synthetic biochemistry and synthetic inorganic chemistry together in a synergistic way. [ABSTRACT FROM AUTHOR]

The reduction of the potent greenhouse gas nitrous oxide requires a catalyst to overcome the large activation energy barrier of this reaction. Its biological decomposition to the inert dinitrogen can be accomplished by denitrifiers through nitrous oxide reductase, the enzyme that catalyzes the last step of the denitrification, a pathway of the biogeochemical nitrogen cycle. Nitrous oxide reductase is a multicopper enzyme containing a mixed valence CuA center that can accept electrons from small electron shuttle proteins, triggering electron flow to the catalytic sulfide-bridged tetranuclear copper “CuZ center”. This enzyme has been isolated with its catalytic center in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S), proven to be spectroscopic and structurally different. In the last decades, it has been a challenge to characterize the properties of this complex enzyme, due to the different oxidation states observed for each of its centers and the heterogeneity of its preparations. The substrate binding site in those two “CuZ center” forms and which is the active form of the enzyme is still a matter of debate. However, in the last years the application of different spectroscopies, together with theoretical calculations have been useful in answering these questions and in identifying intermediate species of the catalytic cycle. An overview of the spectroscopic, kinetics and structural properties of the two forms of the catalytic “CuZ center” is given here, together with the current knowledge on nitrous oxide reduction mechanism by nitrous oxide reductase and its intermediate species. [ABSTRACT FROM AUTHOR]

Background: The COVID- 19 situation in Brazil is complex due to large differences in the shape and size of regional epidemics. Understanding these patterns is crucial to understand future outbreaks of SARS- CoV- 2 or other respiratory pathogens in the country. Methods: We tested 97,950 blood donation samples for IgG antibodies from March 2020 to March 2021 in 8 of Brazil's most populous cities. Residential postal codes were used to obtain representative samples. Weekly age- and sex- specific seroprevalence were estimated by correcting the crude seroprevalence by test sensitivity, specificity, and antibody waning. Results: The inferred attack rate of SARS- CoV- 2 in December 2020, before the Gamma variant of concern (VOC) was dominant, ranged from 19.3% (95% credible interval [CrI] 17.5--21.2%) in Curitiba to 75.0% (95% CrI 70.8--80.3%) in Manaus. Seroprevalence was consistently smaller in women and donors older than 55 years. The age- specific infection fatality rate (IFR) differed between cities and consistently increased with age. The infection hospitalisation rate increased significantly during the Gamma- dominated second wave in Manaus, suggesting increased morbidity of the Gamma VOC compared to previous variants circulating in Manaus. The higher disease penetrance associated with the health system's collapse increased the overall IFR by a minimum factor of 2.91 (95% CrI 2.43--3.53). Conclusions: These results highlight the utility of blood donor serosurveillance to track epidemic maturity and demonstrate demographic and spatial heterogeneity in SARS- CoV- 2 spread. Funding: This work was supported by Itaú Unibanco 'Todos pela Saude' program; FAPESP (grants 18/14389- 0, 2019/21585- 0); Wellcome Trust and Royal Society Sir Henry Dale Fellowship 204311/Z/16/Z; the Gates Foundation (INV- 034540 and INV- 034652); REDS- IV- P (grant HHSN268201100007I); the UK Medical Research Council (MR/S0195/1, MR/V038109/1); CAPES; CNPq (304714/2018- 6); Fundação Faculdade de Medicina; Programa Inova Fiocruz- CE/Funcap - Edital 01/2020 Number: FIO- 0167- 00065.01.00/20 SPU N°06531047/2020; JBS -- Fazer o bem faz bem. [ABSTRACT FROM AUTHOR]

Carbon dioxide accumulation is a major concern for the ecosystems, but its abundance and low cost make it an interesting source for the production of chemical feedstocks and fuels. However, the thermodynamic and kinetic stability of the carbon dioxide molecule makes its activation a challenging task. Studying the chemistry used by nature to functionalize carbon dioxide should be helpful for the development of new efficient (bio)catalysts for atmospheric carbon dioxide utilization. In this work, the ability of Desulfovibrio desulfuricans formate dehydrogenase (Dd FDH) to reduce carbon dioxide was kinetically and mechanistically characterized. The Dd FDH is suggested to be purified in an inactive form that has to be activated through a reduction-dependent mechanism. A kinetic model of a hysteretic enzyme is proposed to interpret and predict the progress curves of the Dd FDH-catalyzed reactions (initial lag phase and subsequent faster phase). Once activated, Dd FDH is able to efficiently catalyze, not only the formate oxidation (kcat of 543 s-1, Km of 57.1 μM), but also the carbon dioxide reduction (kcat of 46.6 s-1, Km of 15.7 μM), in an overall reaction that is thermodynamically and kinetically reversible. Noteworthy, both Dd FDH-catalyzed formate oxidation and carbon dioxide reduction are completely inactivated by cyanide. Current FDH reaction mechanistic proposals are discussed and a different mechanism is here suggested: formate oxidation and carbon dioxide reduction are proposed to proceed through hydride transfer and the sulfo group of the oxidized and reduced molybdenum center, Mo6+=S and Mo4+-SH, are suggested to be the direct hydride acceptor and donor, respectively. [ABSTRACT FROM AUTHOR]

Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd1NiR from Marinobacter hydrocarbonoclasticus (Mhcd1) depends on the presence of its physiological redox partner, cytochrome c552 (cyt c552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd1 and cyt c552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd1 // cyt c552 and Ag // SAM // cyt c552 // Mhcd1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c552 retains its native properties, while the redox potential of apparently intact Mhcd1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd1, reinforcing the idea that subtle and very specific interactions between Mhcd1 and cyt c552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd1. [ABSTRACT FROM AUTHOR]

The prokaryotic formate metabolism is considerably diversified. Prokaryotes use formate in the C1 metabolism, but also evolved to exploit the low reduction potential of formate to derive energy, by coupling its oxidation to the reduction of numerous electron acceptors. To fulfil these varied physiological roles, different types of formate dehydrogenase (FDH) enzymes have evolved to catalyse the reversible 2-electron oxidation of formate to carbon dioxide. This review will highlight our present knowledge about the diverse physiological roles of FDH in prokaryotes, their modular structural organisation and active site structures and the mechanistic strategies followed to accomplish the formate oxidation. In addition, the ability of FDH to catalyse the reverse reaction of carbon dioxide reduction, a potentially relevant reaction for carbon dioxide sequestration, will also be addressed. [ABSTRACT FROM AUTHOR]

There was increase in SIRT-6 ( I P i = 0.015) and SIRT-7 in keratinocytes ( I P i < 0.001); SIRT-6, papillary dermis ( I P i = 0.039); and SIRT-7, glandular epithelium ( I P i = 0.004) and reticular dermis ( I P i = 0.023). Dear Editor, Adults lose 1% of collagen per year because of aging.1 This process impacts negatively more on type-3 (COL3) than type-1 (COL1) collagen and is aggravated by metalloproteinases and photoaging-decreased neocollagenesis.2 Sirtuins (SIRT), a histone-deacetylase family, cause longevity of fibroblasts and regulate collagen synthesis.3 SIRT protect skin against UV-dependent damage by repairing DNA, controlling inflammation, and modulating antioxidant defense.4 SIRT-1, SIRT-3, and SIRT-7 are reduced by UV. Phenol-croton oil peel enhances type-1 and type-3 collagen amounts by stimulating SIRT-6 and SIRT-7. [Extracted from the article]

Abstract: Three fluorinated Mo–Cu–thiolate isomers, [Ph4P]2[S2MoS2Cu(n-SPhF)], [n-SPhF =2-fluorothiophenol (1a)], 3-fluorothiophenol (1b), and 4-fluorothiophenol (1c)] were synthesized and spectroscopically characterized. The 19F-NMR signal of the fluorine atom in the benzene has different chemical shift for each isomer, which is highly influenced by the local environment that can be manipulated by different solvents and solutes. The fluorine-19 chemical shift is an advantageous NMR structural probe in alternative to 1H-NMR [B.K. Maiti, T. Avilés, M. Matzapetakis, I. Moura, S.R. Pauleta, J.J.G. Moura, Eur. J. Inorg. Chem. (2012) 4159.], that can be used to provide local information on the pocket of the metal cluster in the Orange Protein (ORP). [Copyright &y& Elsevier]

Background The United Nation's Program for Reducing Emissions from Deforestation and Forest Degradation (REDD+) aims to reduce the 20% contribution to global emissions of greenhouse gases from the forest sector, offering a financial value of the carbon stored in forests as an incentive for local communities. The pre-requisite for the setup of a participatory REDD + Program is the monitoring, reporting and verification (MRV) of baseline carbon stocks and their changes over time. In this study, we investigated miombo woodland's dynamics in terms of composition, structure and biomass over a 4-year period (2005-2009), and the Carbon Stock Density (CSD) for the year 2009. The study was conducted in the Niassa National Reserve (NNR) in northern Mozambique, which is the 14th largest protected area in the world. Results Mean tree density distributed across 79 species increased slightly between 2005 and 2009, respectively, from 548 to 587 trees ha-1. Julbernardia globiflora (Benth.) was the most important species in this area [importance value index (IVI2005 =61 and IVI2009 = 54)]. The woodlands presented an inverted J-shaped diametric curve, with 69% of the individuals representing the young cohort. Woody biomass had a net increase of 3 Mg ha-1 with the highest growth observed in Dyplorhynchus condilocarpon (Müll.Arg.) Pichon (0.54 Mg ha-1). J. globiflora had a net decrease in biomass of 0.09 Mg ha-1. Total CSD density was estimated at ca. 67 MgC ha-1 ± 24.85 with soils (average 34.72 ± 17.93 MgC ha-1) and woody vegetation (average 29.8 MgC ha-1 ± 13.07) representing the major carbon pools. The results point to a relatively stable ecosystem, but they call for the need to refocus management activities. Conclusions The miombo woodlands in NNR are representative of the woodlands in the eco-region in terms of vegetation structure and composition. They experienced net increase in woody biomass, a considerable recruitment level and low mortality. According to our results, NNR may present good potential for carbon sequestration especially in soils and woody biomass, representing an important potential carbon sink. However, further investigations are needed in order to address the contribution of this area to MRV REDD + initiatives. [ABSTRACT FROM AUTHOR]

Abstract: Nitrous oxide is a potent greenhouse gas, whose atmospheric concentration has been increasing since the introduction of the Haber Bosch process led to the widespread use of nitrogenous fertilizers. One of the pathways to its destruction is reduction to molecular nitrogen by the enzyme nitrous oxide reductase found in denitrifying bacteria. This enzyme catalyzes the last step of the denitrification pathway. It has two copper centers, a binuclear CuA center, similar to the one found in cytochrome c oxidase, and the CuZ center, a unique tetranuclear copper center now known to possess either one or two sulfide bridges. Nitrous oxide reductase has been isolated in different forms, depending on the oxidation state and molecular forms of its Cu centers. Recently, the structure of a purple form, which has both centers in the oxidized state, revealed that the CuZ center has the form [Cu4S2]. This review summarizes the biogenesis and regulation of nitrous oxide reductase, and the spectroscopic and kinetic properties of nitrous oxide reductase. The proposed activation and catalytic mechanism, as well as, electron transfer pathways are discussed in the light of the various structures of the CuZ center. [Copyright &y& Elsevier]

Abstract: The respiratory nitrate reductase complex (NarGHI) from Marinobacter hydrocarbonoclasticus 617 (Mh, formerly Pseudomonas nautica 617) catalyzes the reduction of nitrate to nitrite. This reaction is the first step of the denitrification pathway and is coupled to the quinone pool oxidation and proton translocation to the periplasm, which generates the proton motive force needed for ATP synthesis. The Mh NarGH water-soluble heterodimer has been purified and the kinetic and redox properties have been studied through in-solution enzyme kinetics, protein film voltammetry and spectropotentiometric redox titration. The kinetic parameters of Mh NarGH toward substrates and inhibitors are consistent with those reported for other respiratory nitrate reductases. Protein film voltammetry showed that at least two catalytically distinct forms of the enzyme, which depend on the applied potential, are responsible for substrate reduction. These two forms are affected differentially by the oxidizing substrate, as well as by pH and inhibitors. A new model for the potential dependence of the catalytic efficiency of Nars is proposed. [Copyright &y& Elsevier]

This review focuses on the novel CuZ center of nitrous oxide reductase, an important enzyme owing to the environmental significance of the reaction it catalyzes, reduction of nitrous oxide, and the unusual nature of its catalytic center, named CuZ. The structure of the CuZ center, the unique tetranuclear copper center found in this enzyme, opened a novel area of research in metallobiochemistry. In the last decade, there has been progress in defining the structure of the CuZ center, characterizing the mechanism of nitrous oxide reduction, and identifying intermediates of this reaction. In addition, the determination of the structure of the CuZ center allowed a structural interpretation of the spectroscopic data, which was supported by theoretical calculations. The current knowledge of the structure, function, and spectroscopic characterization of the CuZ center is described here. We would like to stress that although many questions have been answered, the CuZ center remains a scientific challenge, with many hypotheses still being formed. [ABSTRACT FROM AUTHOR]

Pseudoazurins are small type 1 copper proteins that are involved in the flow of electrons between various electron donors and acceptors in the bacterial periplasm, mostly under denitrifying conditions. The previously determined structure of Paracoccus pantotrophus pseudoazurin in the oxidized form was improved to a nominal resolution of 1.4 Å, with R and Rfree values of 0.188 and 0.206, respectively. This high-resolution structure makes it possible to analyze the interactions between the monomers and the solvent structure in detail. Analysis of the high-resolution structure revealed the structural regions that are responsible for monomer-monomer recognition during dimer formation and for protein-protein interaction and that are important for partner recognition. The pseudoazurin structure was compared with other structures of various type 1 copper proteins and these were grouped into families according to similarities in their secondary structure; this may be useful in the annotation of copper proteins in newly sequenced genomes and in the identification of novel copper proteins. [ABSTRACT FROM AUTHOR]

The cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 is able to reduce nitrite to ammonia in a six-electron transfer reaction. Although extensively characterized from the spectroscopic and structural points-of-view, some of its kinetic aspects are still under explored. In this work the kinetic behaviour of ccNiR has been evaluated in a systematic manner using two different spectrophotometric assays carried out in the presence of different redox mediators and a direct electrochemical approach. Solution assays have proved that the specific activity of ccNiR decreases with the reduction potential of the electronic carriers and ammonium is always the main product of nitrite reduction. The catalytic parameters were discussed on the basis of the mediator reducing power and also taking into account the location of their putative docking sites with ccNiR. Due to the fast kinetics of ccNiR, electron delivering from reduced electron donors is rate-limiting in all spectrophotometric assays, so the estimated kinetic constants are apparent only. Nevertheless, this limitation could be overcome by using a direct electrochemical approach which shows that the binding affinity for nitrite decreases whilst turnover increases with the reductive driving force. [ABSTRACT FROM AUTHOR]

Abstract: The paramagnetic effect due to the presence of a metal center with unpaired electrons is no longer considered a hindrance in protein NMR spectroscopy. In the present work, the paramagnetic effect due to the presence of a metal center with unpaired electrons was used to map the interface of an electron transfer complex. Desulfovibrio gigas cytochrome c 3 was chosen as target to study the effect of the paramagnetic probe, Fe-rubredoxin, which produced specific line broadening in the heme IV methyl resonances M21 and M181. The rubredoxin binding surface in the complex with cytochrome c 3 was identified in a heteronuclear 2D NMR titration. The identified heme methyls on cytochrome c 3 are involved in the binding interface of the complex, a result that is in agreement with the predicted complexes obtained by restrained molecular docking, which shows a cluster of possible solutions near heme IV. The use of a paramagnetic probe in 1HNMR titration and the mapping of the complex interface, in combination with a molecular simulation algorithm proved to be a valuable strategy to study electron transfer complexes involving non-heme iron proteins and cytochromes. [Copyright &y& Elsevier]

Abstract: Nitrogen is a vital component in living organisms as it participates in the making of essential biomolecules such as proteins, nucleic acids, etc. In the biosphere, nitrogen cycles between the oxidation states +V and −III producing many species that constitute the biogeochemical cycle of nitrogen. All reductive branches of this cycle involve the conversion of nitrate to nitrite, which is catalyzed by the enzyme nitrate reductase. The characterization of nitrate reductases from prokaryotic organisms has allowed us to gain considerable information on the molecular basis of nitrate reduction. Prokaryotic nitrate reductases are mononuclear Mo-containing enzymes sub-grouped as respiratory nitrate reductases, periplasmic nitrate reductases and assimilatory nitrate reductases. We review here the biological and molecular properties of these three enzymes along with their gene organization and expression, which are necessary to understand the biological processes involved in nitrate reduction. [Copyright &y& Elsevier]

Mononuclear molybdenum and tungsten are found in the active site of a diverse group of enzymes that, in general, catalyze oxygen atom transfer reactions. Enzymes of the xanthine oxidase family are the best-characterized mononuclear Mo-containing enzymes. Several 3D structures of diverse members of this family are known. Recently, the structures of substrate-bound and arsenite-inhibited forms of two members of this family have also been reported. In addition, spectroscopic studies have been utilized to elucidate fine details that complement the structural information. Altogether, these studies have provided an important amount of information on the characteristics of the active site and the electron transfer pathways. [Copyright &y& Elsevier]

Discusses the Mossbauer characterization of the iron-sulfur clusters in Desulfovibrio vulgaris hydrogenase. Reductive activation of aerobically purified D. vulgaris hydrogenase; Oxidation states of the [2Fe][sub H] subcluster; Binding of exogenous CO with H cluster in D. vulgaris hydrogenase.

The kinetics of electron transfer between the Desulfovibro gigas hydrogenase and several electron-transfer proteins from Desulfovibrio species were investigated by cyclic voltammetry, square-wave voltammetry and chronoamperometry. The cytochrome C3 from Desulfovibrio vulgaris (Hildenborough). Desulfovibrio desulfuricans (Norway 4), Desulfovibrio desulfuricans (American Type Culture Collection 27774) and D. gigas (NCIB 9332) were used as redox carriers. They differ in their redox potentials and isoelectric point. Depending on the pH, all the reduced forms of these cytochromes were effective in electron exchange with hydrogenase. Other small electron-transfer proteins such as ferredoxin I, ferredoxin II and rubredoxin from D. gigas were tentatively used redox carriers. Only ferredoxin II was effective in mediating electron exchange between hydrogenase and the working electrode. The second-order rate constants k for the reaction between reduced proteins and hydrogenase were calculated based on the theory of the simplest electrocatalytic mechanism [Moreno, C., Costa, C., Moura, I., Le Gall, J., Liu, M. Y., Payne, W.J., van Dijk, C. & Moura. J.J.G. (1193) Eur. J.. Biochem. 212. 79-86] and the results obtained by cyclic voltammetry were compared with those obtained by chronoamperometry. Values for k of 105-106 M&sup-1; s&sup-1; (cytochrome c1 as electron carrier) and 104M&sup-1; s&sup-1; (ferredoxin II as the electron carrier) were determined. The rate-constant values are discussed in terms of the existence of an electrostatic interaction between the electrode surface and the redox carrier and between the redox carrier and a positively charged part of the enzyme. [ABSTRACT FROM AUTHOR]

The electron-transfer kinetics between three different mediators and the hexahemic enzyme nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774) were investigated by cyclic voltammetry and by chronoamperometry. The mediators, methyl viologen, Desulfovibrio vulgaris (Hildenborough) cytochrome c3 and D. desulfuricans (ATCC 27774) cytochrome c3 differ in structure, redox potential and charge. The reduced form of each mediator exchanged electrons with nitrite reductase. Second-order rate constants, k, were calculated on the basis of the theory for a simple catalytic mechanism and the results, obtained by cyclic voltammetry, were compared with those obtained by chronoamperometry. Values for k are in the range 106-108 M-1 s-1 and increase in the direction D. desulfuricans cytochrome c3→D. vulgaris cytochrome c3→methyl viologen. An explanation is advanced on the basis of electrostatic interactions and relative orientation between the partners involved. Chronoamperometry (computer controlled) offers advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate and better reproducibility). Direct, unmediated electrochemical responses of the hexaheme nitrite reductase were also reported. [ABSTRACT FROM AUTHOR]