The outcome of O-2 activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122)(1) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine)(2)-(carboxylate)(4) ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a mu -1,2-peroxodiiron(III) intermediate,(2 3) which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H-peroxo) in O-2 activation by MMOH.(4) In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the ``extra'' electron that occurs in wt R2 during formation of the formally Fe(LII)Fe(IV) cluster X.(5-7) Decay of the mu1,2-peroxodiiron(III) complex in R2-W38F/D84E gives an initial brown product, which contains very little YI22(.) and which converts very slowly (t(1/2) similar to 7 h) upon incubation at 0 degreesC to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone epsilon -hydroxylation and the resulting phenol has shifted significantly to become st ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with O-16(2) or O-18(2) show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from Or. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Mossbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(LII)-phenolate species is ascribed to a ligand rearrangement in which mu -O2- is lost and the F208-derived phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little epsilon -hydroxyphenylalanine is formed and pathways leading to Y122(.) formation predominate in both R2-D84E and R2-W48F(2-7).

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q Variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical For the catalytic process by controlling the release of the product.

Working teams in enterprise environment are considered as the most advanced forms of work organisation. This means the forms that can improve productivity quality of working life. Nevertheless, it prevails a slow development and dissemination of these advanced organisational forms in European companies. The reason for that lays in a complex linkage factors from social values to the economical pressures. But other factors are also related to the national systems of education training, to the different systems of industrial relations and technology policy.

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The choice over the Portuguese case studies was based on the sample constructed for the application of the firm questionnaires, during the second year of the SOWING project, 1999. This sample was fulfilled of firms among several activity sectors: textile, manufacturing, electronics, transports and software industry, based on NACE – codes (2 – digit level). Thus, we agreed to include in a new database the remaining questionnaires and construct a sample with 113 observations. Concerning the organisational change we make a distinction of three categories of change. First we analyse changes taking place at the inter-firm level (outsourcing, subcontracting, geographic relocation), followed by changes at the organisational level (deconcentration/decentralisation, reduction of hierarchical levels, introduction of cost and profit centres). The third kind of changes analysed will be those taking place at the workplace level (job enlargement/enrichment, changing character of work, work load). The Portuguese studied companies presents a relative uniform pattern considering the variables social competencies, practical knowledge, responsibility and specialized professional qualifications.

The relation between work organisation and technological practices in auto industry is analysed in this article. The concept of “technological practice” in this sector is used to describe the specific ways of embedding information and communication technology applications into the organizational forms and cultural patterns. This concept was developed with the Sowing project (TSER, DG XII) and that approach included either the shop floor co-operation up to the regionally based networks of companies and supporting institutions. The authors studied different sectors in the automotive firms of different European countries (Germany, Belgium and Portugal): shopfloor and production lines, design and management and the local inter-relationships. It was underlined some evidencies of the different alternatives in terms of technological practices for the same sector. Much of the litterature try to disseminate an idea of a single (and optimum) organisational model for the same type of product. And here, even with the same type of technology, and of product (medium-high range), one can find different models, different cultures, different ways of organising the industrial structure (firms, regional institutions, R&D centres) in the same sector (auto industry).

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Mycobacterium tuberculosis KatG is a multifunctional heme enzyme responsible for activation of the antibiotic isoniazid. A KatG(S315T) point mutation is found in >50% of isoniazid-resistant clinical isolates. Since isoniazid activation is thought to involve an oxidation reaction, the redox potential of KatG was determined using cyclic voltammetry, square wave voltammetry, and spectroelectrochemical titrations. Isoniazid activation may proceed via a cytochrome P450-like mechanism. Therefore, the possibility that substrate binding by KatG leads to an increase in the heme redox potential and the possibility that KatG(S315T) confers isoniazid resistance by altering the redox potential were examined. Effects of the heme spin state on the reduction potentials of KatG and KatG(S315T) were also determined. Assessment of the Fe(3+)/Fe(2+) couple gave a midpoint potential of ca. -50 mV for both KatG and KatG(S315T). In contrast to cytochrome P450s, addition of substrate had no significant effect on either the KatG or KatG(S315T) redox potential. Conversion of the heme to a low-spin configuration resulted in a -150 to -200 mV shift of the KatG and KatG(S315T) redox potentials. These results suggest that isoniazid resistance conferred by KatG(S315T) is not mediated through changes in the heme redox potential. The redox potentials of isoniazid were also determined using cyclic and square wave voltammetry, and the results provide evidence that the ferric KatG and KatG(S315T) midpoint potentials are too low to promote isoniazid oxidation without formation of a high-valent enzyme intermediate such as compounds I and II or oxyferrous KatG.

Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa alpha(2) homodimer containing, on average, 0.7 iron atoms/subunit. Mossbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K(3)(Fe(CN)(6)), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A(280)/A(656) ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.

The increasing degree of inter-dependency and integration of economy at a global scale motivated the globalization of scientific and technological knowledge from manufacturing, to the marketing and the strategic management of companies that act as protagonists of these processes. Globalization of markets is inter-connected with intensive incorporation of knowledge in economy. In this sense the continuous evolution of the manufacturing industry structure for an increased high intensity technology elements, namely information and communication technologies, implies that these industrial sub-sectors are conditioning the global performance of economy, the productivity gains and as a consequence the levels of economical and employment growth. This study on “R&D, Innovation and Employment Creation” for the Portuguese Observatory of Employment and Vocational Training (IEFP-MQE) is updating information on the articulation between Job creation, innovation, technology, and R&D. A diagnosis of the state of national S&T research and of processes of technological and organizational innovation was made, as well of the interfacing structures between the knowledge sector and the economical activities. Were made 12 case studies at companies with some innovation activities, interviews to unionists and to Professional associations, and public offices. Was made also a survey to a selected sample of technological infrastructures.

The increasing degree of inter-dependency and integration of economy at a global scale motivated the globalization of scientific and technological knowledge from manufacturing, to the marketing and the strategic management of companies that act as protagonists of these processes. Globalization of markets is inter-connected with intensive incorporation of knowledge in economy. In this sense the continuous evolution of the manufacturing industry structure for an increased high intensity technology elements, namely information and communication technologies, implies that these industrial sub-sectors are conditioning the global performance of economy, the productivity gains and as a consequence the levels of economical and employment growth. This study on “R&D, Innovation and Employment Creation” for the Portuguese Observatory of Employment and Vocational Training (IEFP-MQE) is updating information on the articulation between Job creation, innovation, technology, and R&D. A diagnosis of the state of national S&T research and of processes of technological and organizational innovation was made, as well of the interfacing structures between the knowledge sector and the economical activities. Were made 12 case studies at companies with some innovation activities, interviews to unionists and to Professional associations, and public offices. Was made also a survey to a selected sample of technological infrastructures.

The innovation in the employment behaviour is particularly clear in what concerns the sector composition of the employment changes: the persistent decrease of the industrial employment (even if within a framework of some recovery of the industrial product), a slight expansion in the employment on the tertiary sector, and an important growth of the employment in the construction and public works activities. We can identify then a considerable sector mutation concerning the industrial and the services sectors but also a growth of the atypical forms of employment. Portugal continues not being able to respond to the labour market needs (improvement in the education sector is critical to the catch-up process). The Portuguese cases studied point out to organisational changes supported by ICT, but not determined/induced by it. For most of the changes that were recently developed, ICT had an important role. We didn’t found explicit technological determinism in the relation between ICT and social exclusion.

The innovation in the employment behaviour is particularly clear in what concerns the sector composition of the employment changes: the persistent decrease of the industrial employment (even if within a framework of some recovery of the industrial product), a slight expansion in the employment on the tertiary sector, and an important growth of the employment in the construction and public works activities. We can identify then a considerable sector mutation concerning the industrial and the services sectors but also a growth of the atypical forms of employment. Portugal continues not being able to respond to the labour market needs (improvement in the education sector is critical to the catch-up process). The Portuguese cases studied point out to organisational changes supported by ICT, but not determined/induced by it. For most of the changes that were recently developed, ICT had an important role. We didn’t found explicit technological determinism in the relation between ICT and social exclusion.

The aldehyde oxidoreductase (MOD) isolated from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774) is a member of the xanthine oxidase family of molybdenum-containing enzymes. It has substrate specificity similar to that of the homologous enzyme from Desulfovibrio gigas (MOP) and the primary sequences from both enzymes show 68 % identity. The enzyme was crystallized in space group P6(1)22, with unit cell dimensions of a=b=156.4 A and c=177.1 A, and diffraction data were obtained to beyond 2.8 A. The crystal structure was solved by Patterson search techniques using the coordinates of the D. gigas enzyme. The overall fold of the D. desulfuricans enzyme is very similar to MOP and the few differences are mapped to exposed regions of the molecule. This is reflected in the electrostatic potential surfaces of both homologous enzymes, one exception being the surface potential in a region identifiable as the putative docking site of the physiological electron acceptor. Other essential features of the MOP structure, such as residues of the active-site cavity, are basically conserved in MOD. Two mutations are located in the pocket bearing a chain of catalytically relevant water molecules. As deduced from this work, both these enzymes are very closely related in terms of their sequences as well as 3D structures. The comparison allowed confirmation and establishment of features that are essential for their function; namely, conserved residues in the active-site, catalytically relevant water molecules and recognition of the physiological electron acceptor docking site.

Molybdoenzymes of the xanthine oxidase family contain two [2Fe-2S](1+,2+) clusters that are bound to the protein by very different cysteine motifs. In the X-ray crystal structure of Desulfovibrio gigas aldehyde oxidoreductase, the cluster ligated by a ferredoxin-type motif is close to the protein surface, whereas that ligated by an unusual cysteine motif is in contact with the molybdopterin [Romao, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof, P., and Huber, R. (1995) Science 270, 1170-1176]. These two clusters display distinct electron paramagnetic resonance (EPR) signals: the less anisotropic one, called signal I, is generally similar to the g(av) approximately 1.96-type signals given by ferredoxins, whereas signal II often exhibits anomalous properties such as very large g values, broad lines, and very fast relaxation properties. A detailed comparison of the temperature dependence of the spin-lattice relaxation time and of the intensity of these signals in D. gigas aldehyde oxidoreductase and in milk xanthine oxidase strongly suggests that the peculiar EPR properties of signal II arise from the presence of low-lying excited levels reflecting significant double exchange interactions. The issue raised by the assignment of signals I and II to the two [2Fe-2S](1+) clusters was solved by using the EPR signal of the Mo(V) center as a probe. The temperature dependence of this signal could be quantitatively reproduced by assuming that the Mo(V) center is coupled to the cluster giving signal I in xanthine oxidase as well as in D. gigas aldehyde oxidoreductase. This demonstrates unambiguously that, in both enzymes, signal I arises from the center which is closest to the molybdenum cofactor.

The combination of docking algorithms with NMR data has been developed extensively for the studies of protein-ligand interactions. However, to extend this development for the studies of protein-protein interactions, the intermolecular NOE constraints, which are needed, are more difficult to access. In the present work, we describe a new approach that combines an ab initio docking calculation and the mapping of an interaction site using chemical shift variation analysis. The cytochrome c553-ferredoxin complex is used as a model of numerous electron-transfer complexes. The 15N-labeling of both molecules has been obtained, and the mapping of the interacting site on each partner, respectively, has been done using HSQC experiments. 1H and 15N chemical shift analysis defines the area of both molecules involved in the recognition interface. Models of the complex were generated by an ab initio docking software, the BiGGER program (bimolecular complex generation with global evaluation and ranking). This program generates a population of protein-protein docked geometries ranked by a scoring function, combining relevant stabilization parameters such as geometric complementarity surfaces, electrostatic interactions, desolvation energy, and pairwise affinities of amino acid side chains. We have implemented a new module that includes experimental input (here, NMR mapping of the interacting site) as a filter to select the accurate models. Final structures were energy minimized using the X-PLOR software and then analyzed. The best solution has an interface area (1037.4 A2) falling close to the range of generally observed recognition interfaces, with a distance of 10.0 A between the redox centers.

Nitrous oxide (N20) is a greenhouse gas, the third most significant contributor to global warming. As a key process for N20 elimination from the biosphere, N20 reductases catalyze the two-electron reduction of N20 to N2. These 2 x 65 kDa copper enzymes are thought to contain a CuA electron entry site, similar to that of cytochrome c oxidase, and a CuZ catalytic center. The copper anomalous signal was used to solve the crystal structure of N20 reductase from Pseudomonas nautica by multiwavelength anomalous dispersion, to a resolution of 2.4 A. The structure reveals that the CuZ center belongs to a new type of metal cluster, in which four copper ions are liganded by seven histidine residues. N20 binds to this center via a single copper ion. The remaining copper ions might act as an electron reservoir, assuring a fast electron transfer and avoiding the formation of dead-end products.

A new computationally efficient and automated "soft docking" algorithm is described to assist the prediction of the mode of binding between two proteins, using the three-dimensional structures of the unbound molecules. The method is implemented in a software package called BiGGER (Bimolecular Complex Generation with Global Evaluation and Ranking) and works in two sequential steps: first, the complete 6-dimensional binding spaces of both molecules is systematically searched. A population of candidate protein-protein docked geometries is thus generated and selected on the basis of the geometric complementarity and amino acid pairwise affinities between the two molecular surfaces. Most of the conformational changes observed during protein association are treated in an implicit way and test results are equally satisfactory, regardless of starting from the bound or the unbound forms of known structures of the interacting proteins. In contrast to other methods, the entire molecular surfaces are searched during the simulation, using absolutely no additional information regarding the binding sites. In a second step, an interaction scoring function is used to rank the putative docked structures. The function incorporates interaction terms that are thought to be relevant to the stabilization of protein complexes. These include: geometric complementarity of the surfaces, explicit electrostatic interactions, desolvation energy, and pairwise propensities of the amino acid side chains to contact across the molecular interface. The relative functional contribution of each of these interaction terms to the global scoring function has been empirically adjusted through a neural network optimizer using a learning set of 25 protein-protein complexes of known crystallographic structures. In 22 out of 25 protein-protein complexes tested, near-native docked geometries were found with C(alpha) RMS deviations < or =4.0 A from the experimental structures, of which 14 were found within the 20 top ranking solutions. The program works on widely available personal computers and takes 2 to 8 hours of CPU time to run any of the docking tests herein presented. Finally, the value and limitations of the method for the study of macromolecular interactions, not yet revealed by experimental techniques, are discussed.