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2011
The tetranuclear copper active site of nitrous oxide reductase: the CuZ center, Dell'Acqua, S., Pauleta S. R., Moura I., and Moura J. J. , J Biol Inorg Chem, Feb, Volume 16, Number 2, p.183-94, (2011) AbstractWebsite

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.

Actin as a potential target for decavanadate, Ramos, S., Moura J. J., and Aureliano M. , J Inorg Biochem, Dec, Volume 104, Number 12, p.1234-9, (2011) AbstractWebsite

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 (3.1x10(-3) s(-1)), and an apparent dissociation constant (k(dapp)) of 227.4+/-25.7 muM and 112.3+/-8.7 muM was obtained in absence or presence of 20 muM V(10), respectively. Moreover, concentrations as low as 50 muM 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 muM). 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.

The electron transfer complex between nitrous oxide reductase and its electron donors, Dell'Acqua, S., Moura I., Moura J. J., and Pauleta S. R. , J Biol Inorg Chem, Dec, Volume 16, Number 8, p.1241-54, (2011) AbstractWebsite

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 (N(2)OR), 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 N(2)OR 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 N(2)OR, or an electrostatic nature, in the case of P. denitrificans N(2)OR and A. cycloclastes N(2)OR. A set of well-conserved residues on the N(2)OR surface were identified as being part of the electron transfer pathway from the redox partner to N(2)OR (Ala495, Asp519, Val524, His566 and Leu568 numbered according to the P. nautica N(2)OR sequence). Moreover, we built a model for Wolinella succinogenes N(2)OR, an enzyme that has an additional c-type-heme-containing domain. The structures of the N(2)OR 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 N(2)OR domain is similar to that found in the other electron transfer complexes.

The mechanism of formate oxidation by metal-dependent formate dehydrogenases, Mota, C. S., Rivas M. G., Brondino C. D., Moura I., Moura J. J., Gonzalez P. J., and Cerqueira N. M. , J Biol Inorg Chem, Dec, Volume 16, Number 8, p.1255-68, (2011) AbstractWebsite

Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools. The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species. Our studies indicate that the C-H bond break is an event involved in the rate-limiting step of the catalytic cycle. The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results.

Analysis of the activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase through an electron transfer chain, Paes de Sousa, P. M., Rodrigues D., Timoteo C. G., Simoes Goncalves M. L., Pettigrew G. W., Moura I., Moura J. J., and Correia dos Santos M. M. , J Biol Inorg Chem, Aug, Volume 16, Number 6, p.881-8, (2011) AbstractWebsite

The activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase (CCP) was probed through the mediated electrochemical catalysis by its physiological electron donor, P. stutzeri cytochrome c-551. A comparative study was carried out, by performing assays with the enzyme in the resting oxidized state as well as in the mixed-valence activated form, using cyclic voltammetry and a pyrolytic graphite membrane electrode. In the presence of both the enzyme and hydrogen peroxide, the peak-like signal of cytochrome c-551 is converted into a sigmoidal wave form characteristic of an E(r)C'(i) catalytic mechanism. An intermolecular electron transfer rate constant of (4 +/- 1) x 10(5) M(-1) s(-1) was estimated for both forms of the enzyme, as well as a similar Michaelis-Menten constant. These results show that neither the intermolecular electron transfer nor the catalytic activity is kinetically controlled by the activation mechanism of CCP in the case of the P. stutzeri enzyme. Direct enzyme catalysis using protein film voltammetry was unsuccessful for the analysis of the activation mechanism, since P. stutzeri CCP undergoes an undesirable interaction with the pyrolytic graphite surface. This interaction, previously reported for the Paracoccus pantotrophus CCP, induces the formation of a non-native conformation state of the electron-transferring haem, which has a redox potential 200 mV lower than that of the native state and maintains peroxidatic activity.

Structural redox control in a 7Fe ferredoxin isolated from Desulfovibrio alaskensis, Grazina, R., de Sousa P. M., Brondino C. D., Carepo M. S., Moura I., and Moura J. J. , Bioelectrochemistry, Aug, Volume 82, Number 1, p.22-8, (2011) AbstractWebsite

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) (E(0')=-158+/-5mV); [4Fe-4S](+2/+1) (E(0')=-474+/-5mV) and [3Fe-4S](0/-2) (E(0')=-660+/-5mV). The effect of pH on these signals showed that the reduced [3Fe-4S](0) cluster has a pK'(red)(')=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. 70mV) 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.

Cooperative use of cytochrome cd1 nitrite reductase and its redox partner cytochrome c552 to Improve the selectivity of nitrite biosensing, A.S., Serra, S. Jorge, C. Silveira, J.J.G. Moura, E. Jubete, E. Ochoteco, and G. Almeida M. , Anal Chim Acta, Volume 693, p.41-46, (2011)
Implications of oxidovanadium (IV) binding to actin, Ramos, S., Almeida R. M., Moura J. J., and Aureliano M. , Eur J Inorg Chem, Volume 105, Issue 6, p.777, (2011)
Nitrite biosensing using cytochrome c nitrite reductase: Towards a disposable strip electrode, Correia, C., Rodrigues M., Silveira C. M., Moura J. J. G., Ochoteco E., Jubete E., and Almeida M. G. , Biomedical Engineering Systems and Technologies. Series: Communications in Computer and Information Science, (2011)
Study of membrane ageing and grafting mechanisms using electron paramagnetic resonance, Oliveira, F. R. P., Matos C. T., Moura J. J. G., Portugal C. A. M., and Crespo J. G. , Desalination Water Treatment, Volume 27, p.141–149, (2011)
Cooperative use of cytochrome cd1 nitrite reductase and its redox partner cytochrome c552 to improve the selectivity of nitrite biosensing, Serra, A. S., Jorge S. R., Silveira C. M., Moura J. J. G., Jubete E., Ochoteco E., Cabañero G., Grande H., and Almeida M. G. , Analytica Chimica Acta, Volume 693, Number 1–2, p.41-46, (2011) AbstractWebsite
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2010
DNA damage and metal accumulation in four tissues of feral Octopus vulgaris from two coastal areas in Portugal, Raimundo, Joana, Costa Pedro M., Vale Carlos, Costa Maria Helena, and Moura Isabel , Ecotoxicology and Environmental Safety, Oct, Volume 73, Number 7, p.1543-1547, (2010) AbstractWebsite

The alkaline comet assay has been employed for the first time to estimate the basal DNA damage in the digestive gland, gills, kidney and gonads of Octopus vulgaris. Octopuses were captured in two coastal areas adjacent to the cities of Matosinhos (N) and Olhao (S), Portugal. The area of Matosinhos is influenced by discharges of the Douro River, city of Porto, industries and intensive agriculture, while Olhao is an important fisheries port. Previous works point to contrasting metal availability in the two coastal areas. Among the analysed tissues digestive gland presented the highest levels of Zn, Cu, Cd and Pb. Tissues of specimens from Matosinhos exhibited high levels of Cd and from Olhao enhanced Pb concentrations. The DNA damages in digestive gland, gills and kidney were more accentuated in specimens from Matosinhos than from Olhao, suggesting a stronger effect of contaminants. Elevated strand breakages were registered in digestive gland, recognised for its ability to store and detoxify accumulated metals. The DNA damages in kidney, gills and gonads were lower, reflecting reduced metal accumulation or efficient detoxification. The broad variability of damages in the three tissues may also mirror tissue function, specific defences to genotoxicants and cell-cycle turnover. (C) 2010 Elsevier Inc. All rights reserved.

Association of Zn, Cu, Cd and Pb with protein fractions and sub-cellular partitioning in the digestive gland of Octopus vulgaris living in habitats with different metal levels, Raimundo, J., Vale C., Duarte R., and Moura I. , Chemosphere, Nov, Volume 81, Number 10, p.1314-1319, (2010) AbstractWebsite

Zinc Cu Cd and Pb concentrations were determined in protein fractions of digestive gland and in the whole digestive gland of Octopus vulgaris collected from two areas of the Portuguese coast Approximately 95% of Zn 99% of Cu 85-96% of Cd and 77-86% of Pb were stored in the cytosol suggesting the predominance of cytosolic proteins in the trapping these elements Gel filtration chromatography evidenced the presence of two major groups of proteins with high molecular weight (HMW 144 000-130 000 Da) and low molecular weight (LMW 11 000-6000 Da) The following metal-protein associations were found Zn was distributed between HMW and LMW Cu and Cd in LMW proteins with a minor association with HMW and Pb in HMW proteins The strong positive correlations between Cd Zn and Cu and LMW proteins point to the presence of metalloproteins with high affinity to these elements A shift was registered between the maximum of the ratio 254 280 nm and metal concentrations in the chromatographic profiles This shift may result from metallothioneins having a small participation in the metal binding or protein purification was insufficient and various LMW proteins may be interfering (C) 2010 Elsevier Ltd All rights reserved

An efficient non-mediated amperometric biosensor for nitrite determination, Silveira, C. M., Gomes S. P., Araujo A. N., Montenegro M. C., Todorovic S., Viana A. S., Silva R. J., Moura J. J., and Almeida M. G. , Biosens Bioelectron, May 15, Volume 25, Number 9, p.2026-32, (2010) AbstractWebsite

In this paper we propose the construction of a new non-mediated electrochemical biosensor for nitrite determination in complex samples. The device is based on the stable and selective cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans, which has both high turnover and heterogeneous electron transfer rates. In opposition to previous efforts making use of several redox mediators, in this work we exploited the capacity of ccNiR to display a direct electrochemical response when interacting with pyrolytic graphite (PG) surfaces. To enable the analytical application of such bioelectrode the protein was successfully incorporated within a porous silica glass made by the sol-gel process. In the presence of nitrite, the ccNiR/sol-gel/PG electrode promptly displays catalytic currents indicating that the entrapped ccNiR molecules are reduced via direct electron transfer. This result is noteworthy since the protein molecules are caged inside a non-conductive silica network, in the absence of any mediator species or electron relay. At optimal conditions, the minimum detectable concentration is 120 nM. The biosensor sensitivity is 430 mA M(-1) cm(-2) within a linear range of 0.25-50 microM, keeping a stable response up to two weeks. The analysis of nitrites in freshwaters using the method of standard addition was highly accurated.

Rubredoxin mutant A51C unfolding dynamics: A Forster Resonance Energy Transfer study, Santos, Andrea, Duarte Americo G., Fedorov Alexander, Martinho Jose M. G., and Moura Isabel , Biophysical Chemistry, May, Volume 148, Number 1-3, p.131-137, (2010) AbstractWebsite

The unfolding dynamics of the rubredoxin mutant A51C (RdA51C) from Desulfovibrio vulgaris (DvRd) was studied on the temperature range from 25 degrees C to 90 degrees C and by incubation at 90 degrees C. By Forster Resonance Energy Transfer (FRET) the donor (D; Trp37) to acceptor (A; 1,5-IAEDANS) distance distribution was probed at several temperatures between 25 degrees C and 90 degrees C, and incubation times at 90 degrees C. From 25 degrees C to 50 degrees C the half-width distributions values (hw) are small and the presence of a discrete D-A distance was considered. At temperatures higher than 60 degrees C broader hw values were observed reflecting the existence of a distance distribution. The protein denaturation was only achieved by heating the solution for 2 h at 90 degrees C, as probed by the increase of the D-A mean distance. From Trp fluorescence it was shown that its vicinity was maintained until similar to 70 degrees C, being the protein hydrodynamic radius invariant until 50 degrees C. However, at similar to 70 degrees C a change in the partial unfolding kinetics indicates the disruption of specific H-bonds occurring in the hydrophobic core. The red shift of 13 nm, observed on the Trp37 emission, confirms the exposition of Trp to solvent after protein incubation at 90 degrees C for 2.5 h. (C) 2010 Elsevier B.V. All rights reserved.

An NMR structural study of nickel-substituted rubredoxin, Goodfellow, B. J., Duarte I. C., Macedo A. L., Volkman B. F., Nunes S. G., Moura I., Markley J. L., and Moura J. J. , J Biol Inorg Chem, Mar, Volume 15, Number 3, p.409-20, (2010) AbstractWebsite

The Ni(II) and Zn(II) derivatives of Desulfovibrio vulgaris rubredoxin (DvRd) have been studied by NMR spectroscopy to probe the structure at the metal centre. The beta CH(2) proton pairs from the cysteines that bind the Ni(II) atom have been identified using 1D nuclear Overhauser enhancement (NOE) difference spectra and sequence specifically assigned via NOE correlations to neighbouring protons and by comparison with the published X-ray crystal structure of a Ni(II) derivative of Clostridium pasteurianum rubredoxin. The solution structures of DvRd(Zn) and DvRd(Ni) have been determined and the paramagnetic form refined using pseudocontact shifts. The determination of the magnetic susceptibility anisotropy tensor allowed the contact and pseudocontact contributions to the observed chemical shifts to be obtained. Analysis of the pseudocontact and contact chemical shifts of the cysteine H beta protons and backbone protons close to the metal centre allowed conclusions to be drawn as to the geometry and hydrogen-bonding pattern at the metal binding site. The importance of NH-S hydrogen bonds at the metal centre for the delocalization of electron spin density is confirmed for rubredoxins and can be extrapolated to metal centres in Cu proteins: amicyanin, plastocyanin, stellacyanin, azurin and pseudoazurin.

The 1.4 angstrom resolution structure of Paracoccus pantotrophus pseudoazurin, Najmudin, Shabir, Pauleta Sofia R., Moura Isabel, and Romao Maria J. , Acta Crystallographica Section F-Structural Biology and Crystallization Communications, Jun, Volume 66, p.627-635, (2010) AbstractWebsite

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 angstrom, with R and R(free) 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.

Implications of oxidovanadium(IV) binding to actin, Ramos, S., Almeida R. M., Moura J. J., and Aureliano M. , J Inorg Biochem, Jun, Volume 105, Number 6, p.777-83, (2010) AbstractWebsite

Oxidovanadium(IV), a cationic species (VO(2+)) of vanadium(IV), binds to several proteins, including actin. Upon titration with oxidovanadium(IV), approximately 100% quenching of the intrinsic fluorescence of monomeric actin purified from rabbit skeletal muscle (G-actin) was observed, with a V(50) of 131 muM, whereas for the polymerized form of actin (F-actin) 75% of quenching was obtained and a V(50) value of 320 muM. Stern-Volmer plots were used to estimate an oxidovanadium(IV)-actin dissociation constant, with K(d) of 8.2 muM and 64.1 muM VOSO(4), for G-actin and F-actin, respectively. These studies reveal the presence of a high affinity binding site for oxidovanadium(IV) in actin, producing local conformational changes near the tryptophans most accessible to water in the three-dimensional structure of actin. The actin conformational changes, also confirmed by (1)H NMR, are accompanied by changes in G-actin hydrophobic surface, but not in F-actin. The (1)H NMR spectra of G-actin treated with oxidovanadium(IV) clearly indicates changes in the resonances ascribed to methyl group and aliphatic regions as well as to aromatics and peptide-bond amide region. In parallel, it was verified that oxidovanadium(IV) prevents the G-actin polymerization into F-actin. In the 0-200 muM range, VOSO(4) inhibits 40% of the extent of polymerization with an IC(50) of 15.1 muM, whereas 500 muM VOSO(4) totally suppresses actin polymerization. The data strongly suggest that oxidovanadium(IV) binds to actin at specific binding sites preventing actin polymerization. By affecting actin structure and function, oxidovanadium(IV) might be responsible for many cellular effects described for vanadium.

Relations between mercury, methyl-mercury and selenium in tissues of Octopus vulgaris from the Portuguese Coast, Raimundo, Joana, Vale Carlos, Canario Joao, Branco Vasco, and Moura Isabel , Environmental Pollution, Jun, Volume 158, Number 6, p.2094-2100, (2010) AbstractWebsite

Mercury, methyl-mercury (MeHg) and selenium were determined in digestive gland and mantle of Octopus vulgaris, from three areas of the Portuguese coast. To our knowledge these are the first data on MeHg in cephalopods. Concentrations were higher in the digestive gland and percentage of MeHg in mantle. Enhanced Hg and MeHg levels were obtained in digestive gland of specimens from Olhao (3.1-7.4 and 2.0-5.0 mu g g(-1) respectively). Differences between areas may be partially related to Hg availability. Relationships between concentrations in mantle and digestive gland pointed to proportional increases of Hg and MeHg in tissues of specimens from Matosinhos and Cascais, but relatively constant values in mantle of individuals from Olhao (higher contamination). Se:Hg molar ratio in digestive gland was 32 and 30 in octopus from Matosinhos and Cascais, respectively, and 5.4 from Olhao. The proximity to the unit suggests demethylation as response to elevated MeHg levels in digestive gland. (C) 2010 Elsevier Ltd. All rights reserved.

Enhanced Direct Electron Transfer of a Multihemic Nitrite Reductase on Single-walled Carbon Nanotube Modified Electrodes, Silveira, Celia M., Baur Jessica, Holzinger Michael, Moura Jose J. G., Cosnier Serge, and Gabriela Almeida M. , Electroanalysis, Dec, Volume 22, Number 24, p.2973-2978, (2010) AbstractWebsite

Single-walled carbon nanotubes (SWCNTs) deposits on glassy carbon and pyrolytic graphite electrodes have dramatically enhanced the direct electron transfer of the multihemic nitrite reductase from Desulfovibrio desulfuricans ATCC 27774, enabling a 10-fold increase in catalytic currents. At optimal conditions, the sensitivity to nitrite and the maximum current density were 2.4 +/- 0.1 A L mol(-1) cm(-2) and 1500 mu A cm(-2), respectively. Since the biosensor performance decreased over time, laponite clay and electropolymerized amphiphilic pyrrole were tested as protecting layers. Both coating materials increased substantially the bioelectrode stability, which kept about 90% and 60% of its initial sensitivity to nitrite after 20 and 248 days, respectively.

Metallothioneins and trace elements in digestive gland, gills, kidney and gonads of Octopus vulgaris, Raimundo, J., Costa P. M., Vale C., Costa M. H., and Moura I. , Comparative Biochemistry and Physiology C-Toxicology & Pharmacology, Aug, Volume 152, Number 2, p.139-146, (2010) AbstractWebsite

Metallothionein-like proteins (MT) and V, Cr, Co, Ni, Zn, Cu, As and Cd were determined in digestive gland, gills, kidney and gonads of Octopus vulgaris, from the Portuguese coast. To our knowledge these are the first data on MT in octopus. High concentrations (mu g g(-1), dry mass) of Zn (48050) and Cd (555) were found in digestive gland, and MT reached levels one order of magnitude above the ones registered in wild bivalves. Significantly higher levels of MT in digestive gland and gills of specimens from A and B were in line with elevated Cd concentrations. Principal component analyses (PCA) point to MT-Cd and MT-Cr associations in digestive gland and gills. Despite the high levels of Zn in specimens from B, association with Zn was not obtained. Due to the affinity of MT to various elements, it should not be excluded the possibility of Cd replacing Zn in Zn-MT. Kidney presented higher levels of Cd, Co, Ni and As than gills and gonads, and in the case of As surpassing the levels in digestive gland, but PCA showed no relation with MT. Likewise the MT levels in gonads had no correspondence to the metal concentration variation. (C) 2010 Elsevier Inc. All rights reserved.

A new CuZ active form in the catalytic reduction of N(2)O by nitrous oxide reductase from Pseudomonas nautica, Dell'Acqua, S., Pauleta S. R., Paes de Sousa P. M., Monzani E., Casella L., Moura J. J., and Moura I. , J Biol Inorg Chem, Aug, Volume 15, Number 6, p.967-76, (2010) AbstractWebsite

The final step of bacterial denitrification, the two-electron reduction of N(2)O to N(2), is catalyzed by a multi-copper enzyme named nitrous oxide reductase. The catalytic centre of this enzyme is a tetranuclear copper site called CuZ, unique in biological systems. The in vitro reconstruction of the activity requires a slow activation in the presence of the artificial electron donor, reduced methyl viologen, necessary to reduce CuZ from the resting non-active state (1Cu(II)/3Cu(I)) to the fully reduced state (4Cu(I)), in contrast to the turnover cycle, which is very fast. In the present work, the direct reaction of the activated form of Pseudomonas nautica nitrous oxide reductase with stoichiometric amounts of N(2)O allowed the identification of a new reactive intermediate of the catalytic centre, CuZ degrees , in the turnover cycle, characterized by an intense absorption band at 680 nm. Moreover, the first mediated electrochemical study of Ps. nautica nitrous oxide reductase with its physiological electron donor, cytochrome c-552, was performed. The intermolecular electron transfer was analysed by cyclic voltammetry, under catalytic conditions, and a second-order rate constant of (5.5 +/- 0.9) x 10(5) M(-1 )s(-1) was determined. Both the reaction of stoichiometric amounts of substrate and the electrochemical studies show that the active CuZ degrees species, generated in the absence of reductants, can rearrange to the resting non-active CuZ state. In this light, new aspects of the catalytic and activation/inactivation mechanism of the enzyme are discussed.

Ultrasonic multiprobe as a new tool to overcome the bottleneck of throughput in workflows for protein identification relaying on ultrasonic energy, Santos, H. M., Carreira R., Diniz M. S., Rivas M. G., Lodeiro C., Moura J. J., and Capelo J. L. , Talanta, Apr 15, Volume 81, Number 1-2, p.55-62, (2010) AbstractWebsite

We studied in this work the performance of the new ultrasonic multiprobe in terms of throughput, handling and robustness. The study was conducted using the multiprobe to speed two different proteomics workflows. The "classic" method relaying on overnight protein digestion (12h), was used as the standard procedure. This work clearly shows the importance of testing variables such as ultrasonic amplitude and ultrasonic time when adapting an ultrasonic-based treatment to a new ultrasonic device. The results here presented also shown and confirm the advantage of speed up sample treatment workflows with the aid of ultrasonic energy in combination with a 96-well plate. The methods compared were similar in terms of robustness, but the desalting free method was the fastest, requiring only 2 min/sample for completion. In addition it was also the simplest in terms of handling, since no desalting step was needed. The following standard proteins were successfully identified using the methods studied: bovine serum albumin, alpha-lactalbumin, ovalbumin, carbonic anhydrase, fructose-bisphosphate aldolase A, catalase, chymotrypsinogen A. As case study, the identification of the protein Split-Soret cytochrome c from D. desulfuricans ATCC 27774 was carried out.

Nitrite Biosensing via Selective Enzymes-A Long but Promising Route, Almeida, M. G., Serra A., Silveira C. M., and Moura J. J. , Sensors, Volume 10, Number 12, p.11530-55, (2010) AbstractWebsite

The last decades have witnessed a steady increase of the social and political awareness for the need of monitoring and controlling environmental and industrial processes. In the case of nitrite ion, due to its potential toxicity for human health, the European Union has recently implemented a number of rules to restrict its level in drinking waters and food products. Although several analytical protocols have been proposed for nitrite quantification, none of them enable a reliable and quick analysis of complex samples. An alternative approach relies on the construction of biosensing devices using stable enzymes, with both high activity and specificity for nitrite. In this paper we review the current state-of-the-art in the field of electrochemical and optical biosensors using nitrite reducing enzymes as biorecognition elements and discuss the opportunities and challenges in this emerging market.

Measuring the cytochrome c nitrite reductase activity-practical considerations on the enzyme assays, Silveira, C. M., Besson S., Moura I., Moura J. J., and Almeida M. G. , Bioinorg Chem Appl, (2010) AbstractWebsite

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.