Showing papers by "Isabel Moura published in 2008"

Periplasmic Nitrate Reductase Revisited: A Sulfur Atom Completes the Sixth Coordination of the Catalytic Molybdenum.

Abstract: Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe–4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 A. The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH2 ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (λ = 1.77 A) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S–S distance between the sulfur ligand and the Sγ atom of the molybdenum ligand CysA140 was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 A, indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with 15N-labeled nitrate and in D2O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)–nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.

Electron transfer complex between nitrous oxide reductase and cytochrome c552 from Pseudomonas nautica: kinetic, nuclear magnetic resonance, and docking studies.

Abstract: The multicopper enzyme nitrous oxide reductase (N2OR) catalyzes the final step of denitrification, the two-electron reduction of N2 Ot o N 2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome c552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared (cytochrome vs methylviologen (MV)). In the presence of cytochrome c552, the reaction rate is dependent on the ET reaction and independent of the N2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c552 concentration dependence, we estimate the following kinetic parameters: Kmc552 ) 50.2 ( 9.0 µM and Vmaxc552 ) 1.8 ( 0.6 units/mg. The N2O concentration dependence indicates a KmN2O of 14.0 ( 2.9 µM using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c552 is used as the electron donor (pKa ) 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by 1 H NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c552 is placed near a hydrophobic patch located around the CuA center.

Ecotoxicological assessment of industrial wastewaters in Trancão River Basin (Portugal).

Abstract: It is important to assess the toxicity of complex effluents, since chemical evaluation alone is insufficient to protect the environment. Direct Toxicity Assessment is valuable in the decision process regarding the final disposal of complex wastewaters as it measures the total effects of the discharge, because of its known and unknown chemicals, additionally having some degree of ecological relevance. In Portugal, ecotoxicity tests are not used on a regular basis to control wastewaters. So, an integrated ecotoxicological, physical, and chemical study of wastewaters from 17 industries, in the Trancao River Basin, was carried out viewing proposing a test battery to be used in wastewater evaluation. An approach which does not include an ecotoxicological characterization may not properly evaluate the potential risks of effluent discharges, especially when they are complex. From the study carried out the use of a battery of assays to apply in the monitoring of complex wastewaters was proposed, including Microtox test, Daphnia test, and an algal test. Moreover, the added value of the ecotoxicological assessment of industrial wastewaters was demonstrated and could support the implementation of EU Directives (e.g. IPPC, WFD) within the Portuguese situation.

Enzymatic activity mastered by altering metal coordination spheres.

Abstract: 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.

Influence of the protein staining in the fast ultrasonic sample treatment for protein identification through peptide mass fingerprint and matrix-assisted laser desorption ionization time of flight mass spectrometry.

Abstract: The influence of the protein staining used to visualize protein bands, after in-gel protein separation, for the correct identification of proteins by peptide mass fingerprint (PMF) after application of the ultrasonic in-gel protein protocol was studied. Coomassie brilliant blue and silver nitrate, both visible stains, and the fluorescent dyes Sypro Red and Sypro Orange were evaluated. Results obtained after comparison with the overnight in-gel protocol showed that good results, in terms of protein sequence coverage and number of peptides matched, can be obtained with anyone of the four stains studied. Two minutes of enzymatic digestion time was enough for proteins stained with coomassie blue, while 4 min was necessary when silver or Sypro stainings were employed in order to reach equivalent results to those obtained for the overnigh in-gel protein protocol. For the silver nitrate stain, the concentration of silver present in the staining solution must be 0.09% (w/v) to minimize background in the MALDI mas...

Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

Abstract: Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Ι) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at Em = +197 mV (heme c) and −4.5 mV (heme b). Variable-temperature (4–120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe–4S]+ cluster and overlapping signals associated with at least three types of [4Fe–4S]+ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called “low-pH” and “high-pH,” changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases.

Calcium-Dependent Heme Structure in the Reduced Forms of the Bacterial Cytochrome c Peroxidase from Paracoccus pantotrophus†

Abstract: This work reports for the first time a resonance Raman study of the mixed-valence and fully reduced forms of Paracoccus pantotrophus bacterial cytochrome c peroxidase. The spectra of the active mixed-valence enzyme show changes in the structure of the ferric peroxidatic heme compared to the fully oxidized enzyme; these differences are observed upon reduction of the electron-transferring heme and upon full occupancy of the calcium site. For the mixed-valence form in the absence of Ca2+, the peroxidatic heme is six-coordinate and low-spin on the basis of the frequencies of the structure-sensitive Raman lines: the enzyme is inactive. With added Ca2+, the peroxidatic heme is five-coordinate high-spin and active. The calcium-dependent spectral differences indicate little change in the conformation of the ferrous electron-transferring heme, but substantial changes in the conformation of the ferric peroxidatic heme. Structural changes associated with Ca2+ binding are indicated by spectral differences in the stru...

Benefits of membrane electrodes in the electrochemistry of metalloproteins: mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by horse cytochrome c: a case study

Abstract: A comparative study of direct and mediated electrochemistry of metalloproteins in bulk and membrane-entrapped solutions is presented. This work reports the first electrochemical study of the electron transfer between a bacterial cytochrome c peroxidase and horse heart cytochrome c. The mediated catalysis of the peroxidase was analysed both using the membrane electrode configuration and with all proteins in solution. An apparent Michaelis constant of 66 ± 4 and 42 ± 5 μM was determined at pH 7.0 and 0 M NaCl for membrane and bulk solutions, respectively. The data revealed that maximum activity occurs at 50 mM NaCl, pH 7.0, with intermolecular rate constants of (4.4 ± 0.5) × 106 and (1.0 ± 0.5) × 106 M−1 s−1 for membrane-entrapped and bulk solutions, respectively. The influence of parameters such as pH or ionic strength on the mediated catalytic activity was analysed using this approach, drawing attention to the fact that careful analysis of the results is needed to ensure that no artefacts are introduced by the use of the membrane configuration and/or promoters, and therefore the dependence truly reflects the influence of these parameters on the (mediated) catalysis. From the pH dependence, a pK of 7.5 was estimated for the mediated enzymatic catalysis.

Purification, crystallization and preliminary X-ray diffraction analysis of adenosine triphosphate sulfurylase (ATPS) from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774.

Abstract: Native zinc/cobalt-containing ATP sulfurylase (ATPS; EC 2.7.7.4; MgATP:sulfate adenylyltransferase) from Desulfovibrio desulfuricans ATCC 27774 was purified to homogeneity and crystallized. The orthorhombic crystals diffracted to beyond 2.5 A resolution and the X-ray data collected should allow the determination of the structure of the zinc-bound form of this ATPS. Although previous biochemical studies of this protein indicated the presence of a homotrimer in solution, a dimer was found in the asymmetric unit. Elucidation of this structure will permit a better understanding of the role of the metal in the activity and stability of this family of enzymes.