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).

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 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.

Aldehyde oxidoreductase (AOR) activity has been found in different sulfate reducing organisms (Moura, J. J. G., and Barata, B. A. S. (1994) in Methods in Enzymology (Peck, H. D., Jr., and LeGall, J., Eds.), Vol. 243, Chap. 4. Academic Press; Romao, M. J., Knablein, J., Huber, R., and Moura, J. J. G. (1997) Prog. Biophys. Mol. Biol. 68, 121-144). The enzyme was purified to homogeneity from extracts of Desulfovibrio desulfuricans (Dd) ATCC 27774, a sulfate reducer that can use sulfate or nitrate as terminal respiratory substrates. The protein (AORDd) is described as a homodimer (monomer, circa 100 kDa), contains a Mo-MCD pterin, 2 x [2Fe-2S] clusters, and lacks a flavin group. Visible and EPR spectroscopies indicate a close similarity with the AOR purified from Desulfovibrio gigas (Dg) (Barata, B. A. S., LeGall, J., and Moura, J. J. G. (1993) Biochemistry 32, 11559-11568). Activity and substrate specificity for different aldehydes were determined. EPR studies were performed in native and reduced states of the enzyme and after treatment with ethylene glycol and dithiothreitol. The AORDd was crystallized using ammonium sulfate as precipitant and the crystals belong to the space group P6(1)22, with unit cell dimensions a = b = 156.4 and c = 177.1 A. These crystals diffract to beyond 2.5 A resolution and a full data set was measured on a rotating anode generator. The data were used to solve the structure by Patterson Search methods, using the model of AORDg.

Nitrite reductase from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is a multihaem (type c) membrane-bound enzyme that catalyzes the dissimilatory conversion of nitrite to ammonia. Crystals of the oxidized form of this enzyme were obtained using PEG and CaCl(2) as precipitants in the presence of 3--(decylmethylammonium)propane-1-sulfonate and belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 78.94, b = 104.59, c = 143.18 A. A complete data set to 2.30 A resolution was collected using synchrotron radiation at the ESRF. However, the crystals may diffract to beyond 1.7 A and high-resolution data will be collected in the near future.

The socio-economic subsystem encompassing fisheries may be defined as including not only the harvesting sector but also several related activities occurring both upstream (shipbuilding, gear manufacture) and downstream (processing, distribution and trade). But these closely interrelated economic activities can also be set within a much broader system which would include the ecological, institutional and political influences which frame economic behaviour. The value of this broader conceptualisation is that it treats fisheries not as an isolated and independent economic activity but as part of a more holistic and complex system. This broader perspective is of particular significance when attempting to examine the concept of regional dependence. The socio-economic subsystem for fisheries is dominated by small and medium sized enterprises (SMEs). And Peniche emerges as one of Portugal’s most important fishing ports whether measured in terms of the volume of landings or the total numbers of fishermen. It also has one of the highest levels of fisheries dependence of all coastal municipalities in Portugal with over 20% of its workforce currently engaged in fisheries related employment, faces a daunting and uncertain future. The social fabric of fisheries dependent communities also suffers serious damage; once again, the technocratic approach to management has no solutions to offer. It is essential, therefore, to turn away from the existing approach and to develop instead new forms of intervention; in short, to provide a new vision. This implies change not only to the policy process but also in the attitudes of the social actors and in the preoccupations of fisheries related research. An integrated approach is required based on participative action and the development of an integrated information network.

The socio-economic subsystem encompassing fisheries may be defined as including not only the harvesting sector but also several related activities occurring both upstream (shipbuilding, gear manufacture) and downstream (processing, distribution and trade). But these closely interrelated economic activities can also be set within a much broader system which would include the ecological, institutional and political influences which frame economic behaviour. The value of this broader conceptualisation is that it treats fisheries not as an isolated and independent economic activity but as part of a more holistic and complex system. This broader perspective is of particular significance when attempting to examine the concept of regional dependence. The socio-economic subsystem for fisheries is dominated by small and medium sized enterprises (SMEs). And Peniche emerges as one of Portugal’s most important fishing ports whether measured in terms of the volume of landings or the total numbers of fishermen. It also has one of the highest levels of fisheries dependence of all coastal municipalities in Portugal with over 20% of its workforce currently engaged in fisheries related employment, faces a daunting and uncertain future. The social fabric of fisheries dependent communities also suffers serious damage; once again, the technocratic approach to management has no solutions to offer. It is essential, therefore, to turn away from the existing approach and to develop instead new forms of intervention; in short, to provide a new vision. This implies change not only to the policy process but also in the attitudes of the social actors and in the preoccupations of fisheries related research. An integrated approach is required based on participative action and the development of an integrated information network.

The gene encoding the non-heme iron-containing desulfoferrodoxin from Desulfovibrio vulgaris was cloned in two fragments in order to obtain polypeptides corresponding to the N- and C-terminal domains observed in the tertiary structure. These fragments were expressed in Escherichia coli, purified to homogeneity and biochemically and spectroscopically characterized. Both recombinant fragments behaved as independent metal-binding domains. The N-terminal fragment exhibited properties similar to desulforedoxin, as expected by the presence of a Fe(S-Cys)4 metal binding motif. The C-terminal fragment, which accommodates a Fe(Nepsilon-His)3(Ndelta-His)(S-Cys) center, was shown to have properties similar to neelaredoxin, except for the reaction with superoxide. The activities of desulfoferrodoxin and of the expressed C-terminal fragment were tested with superoxide in the presence and absence of cytochrome c. The results are consistent with superoxide reductase activity and a possible explanation for the low superoxide consumption in the superoxide dismutase activity assays is proposed.

The enthalpy and entropy changes associated with protein reduction (deltaHdegrees,(rc), deltaSdegrees,(rc)) were determined for a number of low-potential iron-sulfur proteins through variable temperature direct electrochemical experiments. These data add to previous estimates making available, overall, the reduction thermodynamics for twenty species from various sources containing all the different types of metal centers. These parameters are discussed with reference to structural data and calculated electrostatic metal-environment interaction energies, and redox properties of model complexes. This work, which is the first systematic investigation on the reduction thermodynamics of Fe-S proteins, contributes to the comprehension of the determinants of the differences in reduction potential among different protein families within a novel perspective. Moreover, comparison with analogous data obtained previously for electron transport (ET) metalloproteins with positive reduction potentials, i.e., cytochromes c, blue copper proteins, and HiPIPs, helps our understanding of the factors controlling the reduction potential in ET species containing different metal cofactors. The main results of this work can be summarized as follows.

The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named Cu(A) and Cu(Z). Cu(Z) could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)=2.015, A(x)=1.5 mT, g(y)=2.071, A(y)=2 mT, g(z)=2.138, A(z)=7 mT) and a strong absorption at approximately 640 nm. Cu(A) can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x)=g(y)= 2.021, A(x) = A(y)=0 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center. In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+)-Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)). In both forms, Cu(Z) remains reduced (S = 1/2). Complete crystallographic data at 2.4 A indicate that Cu(A) is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu(Z) is a novel tetracopper cluster [Brown, K., et al. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.

The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named CUA and Cut. Cut could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)= 2.015, A(x) = 1.5 mT, g(y) = 2.071, A(y) = 2 mT, g(z) = 2.138, A(z) = 7 mT) and a strong absorption at similar to 640 nm. Cu-A can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x) g(y) = 2.021, A(x) = A(y) = 0 T, g(z) =0.178, A(z) = 4 mT) and absorption bands at 480, 540, and similar to 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the CuA center. In form A, CUA is predominantly oxidized (S = 1/2, Cu1.5+-Cu1.5+), while in form B it is mostly in the one-electron reduced state (S = 0, Cu1+-Cu1+). In both forms, Cu-Z remains reduced (S = 1/2). Complete crystallographic data at 2.4 Angstrom indicate that Cu-A is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu-Z is a novel tetracopper cluster [Brown, K., et ai. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.

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 Fe3+/Fe2+ 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 IT or oxyferrous KatG.