Cytochrome c peroxidase (CCP) catalyses the reduction of H2O2 to H2O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca2+ and was refined at 2.2 Angstrom resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca2+ dependent and was crystallized at pH 5.3 and refined at 2.4 Angstrom resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase.

A novel adenylate kinase (AK) has recently been purified from Desulfovibrio gigas and characterized as a Co(2+)/Zn(2+)-containing enzyme: this is an unusual characteristic for AKs from Gram-negative bacteria, in which these enzymes are normally devoid of metals. Here, we studied the conformational stability of holo- and apo-AK as a function of temperature by differential scanning calorimetry (DSC), circular dichroism (CD), and intrinsic fluorescence spectroscopy. The thermal unfolding of AK is a cooperative two-state process, and is sufficiently reversible in the 9-11 pH range, that can be correctly interpreted in terms of a simple two-state thermodynamic model. The spectral parameters as monitored by ellipticity changes in the CD spectra of the enzyme as well as the decrease in tryptophan intensity emission upon heating were seen to be good complements to the highly sensitive but integral DSC-method.

Cytochrome c peroxidase (CCP) catalyses the reduction of H2O2 to H2O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca2+ and was refined at 2.2 Angstrom resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca2+ dependent and was crystallized at pH 5.3 and refined at 2.4 Angstrom resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase.

The synthesis, protonation behavior, and Cu2+ and Zn2+ coordination chemistry of the novel bibrachial aza lariat ether (naphthalen-1-ylmethyl)[2-(20-{2-[(naphthalen-1-ylmethyl)amino]ethyl}-3,6,9,17,20,23,29,30-octaazatricyclo-[23.3.1.1*11,15*]triaconta-1(29),11(30),12,14,25,27-hexaen-6-yl)ethyl]amine (L) are discussed. The macrocycle, which has two aminoethyl naphthyl moieties symmetrically appended to a 2:2 azapyridinophane structure, displays, in the pH range 2-11, six protonation steps that correspond to the protonation of the secondary amino groups. Steady-state fluorescence measurements show emissions due to the monomer and to the excimer formed between the two naphthalene fragments of the macrocycle. The time-resolved fluorescence data, obtained by the time-correlated single photon counting technique, show that a significant percentage of excimer is preformed as ground-state dimers. The ligand L forms with the metal ions Cu2+ and Zn2+ mono- and dinuclear complexes in aqueous solution. The influence of metal coordination in the fluorescence emission of L is analyzed. The acid-base, coordination capabilities, and emissive behavior of L are compared with those presented by its synthetic precursor L1, which has a tripodal tris(2-aminoethyl)amine structure functionalized at one of its terminal amino groups with a naphthyl moiety.

The structure of a novel c7-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 Å resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c7-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c7 molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, Eapp, of domain C is −105 mV, 50 mV higher than that of PpcA.

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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 H-1 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. (C) 2003 Elsevier B.V. All rights reserved.

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The fluorescence emission properties of a series of chemosensors containing a polyamine receptor bearing an anthracene signaling unit were studied. The fluorescence emission intensity is dependent on the protonation degree of the receptor, the fully protonated form exhibiting the highest emission intensity. By removing protons from the nitrogens a quenching effect can be observed, due to an electron-transfer from the amine to the excited fluorophore. The rate constant of the quenching process is exponentially dependent on the distance of the nitrogen from which the electron is transferred (beta = 0.6Angstrom(-1)). The ability of the chemosensors for signaling anions was tested through the model anion hexacyanocobaltate(III). The temperature dependence of the association constants shows that at least for this compound, the change in solvation entropy is probably the controlling parameter to account for the binding. (C) 2003 Elsevier Science B.V. All rights reserved.

Spectroscopic methods combined with density functional calculations are used to develop a detailed bonding description of the mu(4)-sulfide bridged tetranuclear Cu(Z) cluster in N(2)O reductase. The ground state of Cu(Z) has the 1Cu(II)/3Cu(I) configuration. The single electron hole dominantly resides on one Cu atom (Cu(I)) and partially delocalizes onto a second Cu atom (Cu(II)) via a Cu(I)-S-Cu(II) sigma/sigma superexchange pathway which is manifested by a Cu(II) --> Cu(I) intervalence transfer transition in absorption. The observed excited-state spectral features of Cu(Z) are dominated by the S --> Cu(I) charge-transfer transitions and Cu(I) based d-d transitions. The intensity pattern of individual S --> Cu(I) charge-transfer transitions reflects different bonding interactions of the sulfur valence orbitals with the four Cu's in the Cu(Z) cluster, which are consistent with the individual Cu-S force constants obtained from a normal coordinate analysis of the Cu(Z) resonance Raman frequencies and profiles. The Cu(I) d orbital splitting pattern correlates with its distorted T-shaped ligand field geometry and accounts for the observed low g( parallel ) value of Cu(Z) in EPR. The dominantly localized electronic structure description of the Cu(Z) site results from interactions of Cu(II) with the two additional Cu's of the cluster (Cu(III)/Cu(IV)), where the Cu-Cu electrostatic interactions lead to hole localization with no metal-metal bonding. The substrate binding edge of Cu(Z) has a dominantly oxidized Cu(I) and a dominantly reduced Cu(IV). The electronic structure description of Cu(Z) provides a strategy to overcome the reaction barrier of N(2)O reduction at this Cu(I)/Cu(IV) edge by simultaneous two-electron transfer to N(2)O in a bridged binding mode. One electron can be donated directly from Cu(IV) and the other from Cu(II) through the Cu(II)-S-Cu(I) sigma/sigma superexchange pathway. A frontier orbital scheme provides molecular insight into the catalytic mechanism of N(2)O reduction by the Cu(Z) cluster.

A propose for this article is to present information on the emergence of the knowledge field of sociology of work and labor studies in Portugal. The period of 1974-1984 produced an interesting bibliography in the fields of social stratification, labor conflicts and social history. However, and since the mid-1980s, significant changes have emerged. There took place an increased theoretical diversity and also there was a major preoccupation with the development of a more professional teaching of the discipline. At this time, the first graduated specialists in Sociology appeared in the labor market since 1980, working in municipalities, industrial firms, public health institutions, schools, or even in research and development (R&D) units. The Portuguese association in this field (APSIOT) organized many scientific meetings, debates with unionists, managers and politicians, beside the regular publication of review “Organiza{\c c}ões e Trabalho” (Organization and Work) since 1989. It started the diffusion of a professional culture of sociologists who associate science and their occupation concerning themselves not only with scientific quality, but also with technical competence and social responsibility. At the same time, one could assist to the participation of sociologists in the social and organizational molding of new technologies in order to promote alternative production systems.

Intramolecular electron (eT) and energy transfer (ET) have shown to occur in a covalently linked donor-acceptor (CLDA) system consisting of a naphthalene donor covalently linked through a polyamine chain connector to an anthracene acceptor; the connector has been chosen in order to switch ON or OFF the energy flux as a function of its protonation state as well as by co-ordination to Zn2+. The largest energy transfer efficiency (eta = 0.61) occurs for the fully protonated form (pH < 2), while at pH > 9 (eT) from the lone pairs of the nitrogens to the excited fluorophore takes place, leading to complete quenching of the emission. On the other hand at neutral and basic pH values. coordination of Zn2+ prevents the eT quenching allowing the ET process to occur. (C) 2002 Elsevier Science B.V. All rights reserved.

The catalytic step that initiates formation of the ferric oxy-hydroxide mineral core in the central cavity of H-type ferritin involves rapid oxidation of ferrous ion by molecular oxygen (ferroxidase reaction) at a binuclear site (ferroxidase site) found in each of the 24 subunits. Previous investigators have shown that the first detectable reaction intermediate of the ferroxidase reaction is a diferric-peroxo intermediate, F-peroxo, formed within 25 ms, which then leads to the release of H2O2 and formation of ferric mineral precursors. The stoichiometric relationship between F-peroxo, H2O2, and ferric mineral precursors, crucial to defining the reaction pathway and mechanism, has now been determined. To this end, a horseradish peroxidase-catalyzed spectrophotometric method was used as an assay for H2O2. By rapidly mixing apo M ferritin from frog, Fe2+, and O-2 and allowing the reaction to proceed for 70 ms when F-peroxo has reached its maximum accumulation, followed by spraying the reaction mixture into the H2O2 assay solution, we were able to quantitatively determine the amount of H2O2 produced during the decay of F-peroxo. The correlation between the amount of H2O2 released with the amount of F-peroxo accumulated at 70 ms determined by Mossbauer spectroscopy showed that F-peroxo decays into H2O2 with a stoichiometry of 1 F-peroxo:H2O2. When the decay of F-peroxo was monitored by rapid freeze-quench Mossbauer spectroscopy, multiple diferric mu-oxo/mu-hydroxo complexes and small polynuclear ferric clusters were found to form at rate constants identical to the decay rate of F-peroxo. This observed parallel formation of multiple products (H2O2, diferric complexes, and small polynuclear clusters) from the decay of a single precursor (F-peroxo) provides useful mechanistic insights into ferritin mineralization and demonstrates a flexible ferroxidase site.

Superoxide reductases have now been well characterized from several organisms. Unique biochemical features include the ability of the reduced enzyme to react with O2- but not dioxygen (reduced SORs are stable in an aerobic atmosphere for hours). Future biochemical assays that measure the reaction of SOR with O2- should take into account the difficulties of assaying O2- directly and the myriad of redox reactions that can take place between components in the assay, for example, direct electron transfer between cytochrome c and Dfx. Future prospects include further delineation of the reaction mechanisms, characterization of the putative (hydro)peroxo intermediate, and studies that uncover the components between reduced pyridine nucleotides and SOR in the metabolic pathway responsible for O2- detoxification.