According to the model proposed in previous papers [Pettigrew, G. W., Prazeres, S., Costa, C., Palma, N., Krippahl, L., and Moura, J. J. (1999) The structure of an electron-transfer complex containing a cytochrome c and a peroxidase, J. Biol. Chem. 274, 11383-11389; Pettigrew, G. W., Goodhew, C. F., Cooper, A., Nutley, M., Jumel, K., and Harding, S. E. (2003) Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans, Biochemistry 42, 2046-2055], cytochrome c peroxidase of Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550) at different sites on its molecular surface. Here we use (1)H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome c(550) bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550) show that the enzyme can accommodate both cytochromes at the same time on adjacent sites. Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome c in the other site. The presence of a substantial capture surface for small cytochromes on the cytochrome c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently.

The cytochrome c nitrite reductase is isolated from the membranes of the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 as a heterooligomeric complex composed by two subunits (61 kDa and 19 kDa) containing c-type hemes, encoded by the genes nrfA and nrfH, respectively. The extracted complex has in average a 2NrfA:1NrfH composition. The separation of ccNiR subunits from one another is accomplished by gel filtration chromatography in the presence of SDS. The amino-acid sequence and biochemical subunits characterization show that NrfA contains five hemes and NrfH four hemes. These considerations enabled the revision of a vast amount of existing spectroscopic data on the NrfHA complex that was not originally well interpreted due to the lack of knowledge on the heme content and the oligomeric enzyme status. Based on EPR and Mossbauer parameters and their correlation to structural information recently obtained from X-ray crystallography on the NrfA structure [Cunha, C.A., Macieira, S., Dias, J.M., Almeida, M.G., Goncalves, L.M.L., Costa, C., Lampreia, J., Huber, R., Moura, J.J.G., Moura, I. & Romao, M. (2003) J. Biol. Chem. 278, 17455-17465], we propose the full assignment of midpoint reduction potentials values to the individual hemes. NrfA contains the high-spin catalytic site (-80 mV) as well as a quite unusual high reduction potential (+150 mV)/low-spin bis-His coordinated heme, considered to be the site where electrons enter. In addition, the reassessment of the spectroscopic data allowed the first partial spectroscopic characterization of the NrfH subunit. The four NrfH hemes are all in a low-spin state (S = 1/2). One of them has a gmax at 3.55, characteristic of bis-histidinyl iron ligands in a noncoplanar arrangement, and has a positive reduction potential.

The gene encoding cytochrome c nitrite reductase (NrfA) from Desulfovibrio desulfuricans ATCC 27774 was sequenced and the crystal structure of the enzyme was determined to 2.3-A resolution. In comparison with homologous structures, it presents structural differences mainly located at the regions surrounding the putative substrate inlet and product outlet, and includes a well defined second calcium site with octahedral geometry, coordinated to propionates of hemes 3 and 4, and caged by a loop non-existent in the previous structures. The highly negative electrostatic potential in the environment around hemes 3 and 4 suggests that the main role of this calcium ion may not be electrostatic but structural, namely in the stabilization of the conformation of the additional loop that cages it and influences the solvent accessibility of heme 4. The NrfA active site is similar to that of peroxidases with a nearby calcium site at the heme distal side nearly in the same location as occurs in the class II and class III peroxidases. This fact suggests that the calcium ion at the distal side of the active site in the NrfA enzymes may have a similar physiological role to that reported for the peroxidases.

Metallothioneins (MT) were obtained after purification from metal-exposed clams (Ruditapes decussatus) using gel-permeation and ion-exchange chromatography. Four cadmium-metallothioneins (CdMTs) were resolved by ion-exchange chromatography and they all had similar molecular weights, high cadmium content and an absorption spectra indicative of the presence of characteristic Cd-S aggregates. The NH(2)-terminal sequence suggests the presence of at least two class I clam MT isoforms. For the other two putative clam CdMTs isolated, the results of the amino acid determination were inconclusive. One was slightly contaminated and the other one had a blocked NH(2)-terminal. These clam metalothioneins contain glycine, which seems to be a common feature of molluscan MT family and exhibited more similarity to oysters than to mussels. Further investigation on the inducibility of these isoforms will be necessary if clams are to be used as biomarkers of metal exposure.

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A CMOS analogue circuit for Gaussian noise generation as well as a novel circuit for transforming Gaussian noise into uniform noise, both designed for operating with a supply voltage of 1.5V, are presented. Both circuits are optimized for a 0.35 {\ensuremathμ}m standard CMOS technology using an equation-based design methodology based on genetic algorithms. Electrical simulations demonstrate that high noise amplitudes together with reasonable bandwidths can be achieved with relatively low power dissipation. Potential applications include self-calibration and on-chip self-testing of video-rate analogue-to-digital converters.

A CMOS analogue circuit for Gaussian noise generation as well as a novel circuit for transforming Gaussian noise into uniform noise, both designed for operating with a supply voltage of 1.5V, are presented. Both circuits are optimized for a 0.35 {\ensuremathμ}m standard CMOS technology using an equation-based design methodology based on genetic algorithms. Electrical simulations demonstrate that high noise amplitudes together with reasonable bandwidths can be achieved with relatively low power dissipation. Potential applications include self-calibration and on-chip self-testing of video-rate analogue-to-digital converters.

Bax is a potent pro-apoptotic member of the Bcl-2 protein family that localizes to the mitochondrial membrane during apoptosis. Tauroursodeoxycholic acid (TUDCA) modulates the apoptotic threshold, in part, by preventing Bax translocation both in vitro and in vivo. The mechanisms by which Bax induces and TUDCA inhibits release of cytochrome c are unclear. We show here that recombinant Bax protein induced cytochrome c release in isolated mitochondria without detectable swelling. Co-incubation with TUDCA prevented efflux of mitochondrial factors and proteolytic processing of caspases in cytosolic extracts. Spectroscopic analyses of mitochondria exposed to Bax revealed increased polarity and fluidity of the membrane lipid core as well as altered protein order, indicative of Bax binding, together with loss of spin-label paramagnetism, characteristic of oxidative damage. TUDCA markedly abrogated the Bax-induced membrane perturbation. In conclusion, our results indicate that Bax protein directly induces cytochrome c release from mitochondria through a mechanism that does not require the permeability transition. Rather, it is accompanied by changes in the organization of membrane lipids and proteins. TUDCA is a potent inhibitor of Bax association with mitochondria. Thus, TUDCA modulates apoptosis by suppressing mitochondrial membrane perturbation through pathways that are also independent of the mitochondrial permeability transition.

A lot has been written over the last decade with regard to Toyota and the productive model associated to it (toyota-ism). And more specifically concerning the "(…) best-seller that changed the... sociological world" (Castillo, 1998: 31). But the case of Salvador Caetano’s Ovar Industrial Division (OID), that assembles Toyota light commercial vehicles in Portugal, allows us to put forward a sub-hypothesis that fits into the analysis schema proposed in the First GERPISA International Program – "In short, GERPISA members considered that the plurality of models was much a plausible hypothesis deserving testing as that of the diffusion of a unique model (…)" (Boyer, Freyssenet, 2001: 42). So we add: and within Toyota itself, is it not true that different productive models co-exist – especially when delocalised – depending, amongst other factors, on the degree of Toyota participation – in terms of capital and technology transfer – in the local company (strong or weak) and on the markets to be reached (internal or external)? If so, what work system can we expect to find in a plant that presents such peculiar characteristics as this one?

A lot has been written over the last decade with regard to Toyota and the productive model associated to it (toyota-ism). And more specifically concerning the "(…) best-seller that changed the... sociological world" (Castillo, 1998: 31). But the case of Salvador Caetano’s Ovar Industrial Division (OID), that assembles Toyota light commercial vehicles in Portugal, allows us to put forward a sub-hypothesis that fits into the analysis schema proposed in the First GERPISA International Program – "In short, GERPISA members considered that the plurality of models was much a plausible hypothesis deserving testing as that of the diffusion of a unique model (…)" (Boyer, Freyssenet, 2001: 42). So we add: and within Toyota itself, is it not true that different productive models co-exist – especially when delocalised – depending, amongst other factors, on the degree of Toyota participation – in terms of capital and technology transfer – in the local company (strong or weak) and on the markets to be reached (internal or external)? If so, what work system can we expect to find in a plant that presents such peculiar characteristics as this one?

This article describes the method and results of our participation in the Critical Assessment of PRediction of Interactions (CAPRI) experiment, using the protein docking program BiGGER (Bimolecular complex Generation with Global Evaluation and Ranking) (Palma et al., Proteins 2000;39:372-384). Of five target complexes (CAPRI targets 2, 4, 5, 6, and 7), only one was successfully predicted (target 6), but BiGGER generated reasonable models for targets 4, 5, and 7, which could have been identified if additional biochemical information had been available.

Membrane electrodes (ME) were constructed using gold, glassy carbon and pyrolytic graphite supports and a dialysis membrane, and used to study the electrochemical behavior of small size electron transfer proteins: monohemic cytochrome c(522) from Pseudomonas nautica and cytochrome c(533) as well as rubredoxin from Desulfovibrio vulgaris. Different electrochemical techniques were used including cyclic voltammetry (CV), square wave voltammetry (SW) and differential pulse voltammetry (DP). A direct electrochemical response was obtained in all cases except with rubredoxin where a facilitator was added to the protein solution entrapped between the membrane and the electrode surface. Formal potentials and heterogeneous charge transfer rate constants were determined from the voltammetric data. The influence of the ionic strength and the pH of the medium on the electrochemical response at the ME were analyzed. The benefits from the use of the ME in protein electrochemistry and its role in modulating the redox behavior are analyzed. A critical comparison is presented with data obtained at non-MEs. Finally, the interactions that must be established between the proteins and the electrode surfaces are discussed, thereby modeling molecular interactions that occur in biological systems. (C) 2002 Elsevier Science B.V. All rights reserved.

The differences in geometry at the metal centres in the two known [Fe-4S] proteins rubredoxin (Rd) and desulforedoxin (Dx) are postulated to be a result of the different spacing of the C-terminal cysteine pair in the two proteins. In order to address this question, two mutants of Desulfovibrio gigas Dx with modified cysteinyl spacing were prepared and their solution structures have been determined by NMR. Mutant 1 of Dx (DxM1) has a single glycine inserted between the adjacent cysteines (C28 and C29) found in the wild type Dx sequence. Mutant 3 (DxM3) has two amino acid residues, -P-V-, inserted between C28 and C29 in order to mimic the primary sequence found in Rd from Desulfovibrio gigas. The solution structure of DxM1 exists, like wild type Dx, as a dimer in solution although the single glycine inserted between the adjacent cysteines disrupts the stability of the dimer resulting in exchange between a dimer state and a small population of another, probably monomeric, state. For DxM3 the two amino acid residues inserted between the adjacent cysteines results in a monomeric protein that has a global fold near the metal centre very similar to that found in Rd.

The production of cytochrome c peroxidase (CCP) from Pseudomonas (Ps.) stutzeri (ATCC 11607) was optimized by adjusting the composition of the growth medium and aeration of the culture. The protein was isolated and characterized biochemically and spectroscopically in the oxidized and mixed valence forms. The activity of Ps. stutzeri CCP was studied using two different ferrocytochromes as electron donors: Ps. stutzeri cytochrome C-551 (the physiological electron donor) and horse heart cytochrome c. These electron donors interact differently with Ps. stutzeri CCP, exhibiting different ionic strength dependence. The CCP from Paracoccus (Pa.) denitrificans was proposed to have two different Ca2+ binding sites: one usually occupied (site I) and the other either empty or partially occupied in the oxidized enzyme (site II). The Ps. stutzeri enzyme was purified in a form with tightly bound Ca2+. The affinity for Ca2+ in the mixed valence enzyme is so high that Ca2+ returns to it from the EGTA which was added to empty the site in the oxidized enzyme. Molecular mass determination by ultracentrifugation and behavior on gel filtration chromatography have revealed that this CCP is isolated as an active dimer, in contrast to the Pa. denitrificans CCP which requires added Ca2+ for formation of the dimer and also for activation of the enzyme. This is consistent with the proposal that Ca2+ in the bacterial peroxidases influences the monomer/dimer equilibrium and the transition to the active form of the enzyme. Additional Ca2+ does affect both the kinetics of oxidation of horse heart cytochrome c (but not cytochrome C-551) and higher aggregation states of the enzyme. This suggests the presence of a superficial Ca2+ binding site of low affinity.

Superoxide reductases catalyze the monovalent reduction of superoxide anion to hydrogen peroxide. Spectroscopic evidence for the formation of a dinuclear cyano-bridged adduct after K(3)Fe(CN)(6) oxidation of the superoxide reductases neelaredoxin from Treponema pallidum and desulfoferrodoxin from Desulfovibrio vulgaris was reported. Oxidation with K(3)Fe(CN)(6) reveals a band in the near-IR with lambda(max) at 1020 nm, coupled with an increase of the iron content by almost 2-fold. Fourier transform infrared spectroscopy provided additional evidence with CN-stretching vibrations at 2095, 2025-2030, and 2047 cm(-)(1), assigned to a ferrocyanide adduct of the enzyme. Interestingly, the low-temperature electronic paramagnetic resonance (EPR) spectra of oxidized TpNlr reveal at least three different species indicating structural heterogeneity in the coordination environment of the active site Fe ion. Given the likely 6-coordinate geometry of the active site Fe(3+) ion in the ferrocyanide adduct, we propose that the rhombic EPR species can serve as a model of a hexacoordinate form of the active site.

The production of cytochrome c peroxidase (CCP) from Pseudomonas (Ps.) stutzeri (ATCC 11607) was optimized by adjusting the composition of the growth medium and aeration of the culture. The protein was isolated and characterized biochemically and spectroscopically in the oxidized and mixed valence forms. The activity of Ps. stutzeri CCP was studied using two different ferrocytochromes as electron donors: Ps. stutzeri cytochrome C-551 (the physiological electron donor) and horse heart cytochrome c. These electron donors interact differently with Ps. stutzeri CCP, exhibiting different ionic strength dependence. The CCP from Paracoccus (Pa.) denitrificans was proposed to have two different Ca2+ binding sites: one usually occupied (site I) and the other either empty or partially occupied in the oxidized enzyme (site II). The Ps. stutzeri enzyme was purified in a form with tightly bound Ca2+. The affinity for Ca2+ in the mixed valence enzyme is so high that Ca2+ returns to it from the EGTA which was added to empty the site in the oxidized enzyme. Molecular mass determination by ultracentrifugation and behavior on gel filtration chromatography have revealed that this CCP is isolated as an active dimer, in contrast to the Pa. denitrificans CCP which requires added Ca2+ for formation of the dimer and also for activation of the enzyme. This is consistent with the proposal that Ca2+ in the bacterial peroxidases influences the monomer/dimer equilibrium and the transition to the active form of the enzyme. Additional Ca2+ does affect both the kinetics of oxidation of horse heart cytochrome c (but not cytochrome C-551) and higher aggregation states of the enzyme. This suggests the presence of a superficial Ca2+ binding site of low affinity.

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.

The tetranuclear CuZ cluster catalyzes the two-electron reduction of N2O to N2 and H2O in the enzyme nitrous oxide reductase. This study shows that the fully reduced 4CuI form of the cluster correlates with the catalytic activity of the enzyme. This is the first demonstration that the S = 1/2 form of CuZ can be further reduced. Complementary DFT calculations support the experimental findings and demonstrate that N2O binding in a bent mu-1,3-bridging mode to the 4CuI form is most efficient due to strong back-bonding from two reduced copper atoms. This back-donation activates N2O for electrophilic attack by a proton.

Pulmonary hypertension is a significant problem to take into account in the post-operative management of cardiac patients, especially valvular patients. Inhaled nitric oxide allows more effective control of pulmonary pressure and other hemodynamic parameters, with better post-operative results. We present a clinical case of a patient with mitral stenosis and severe pulmonary hypertension, with post-operative hemodynamic instability, in which we used inhaled nitric oxide for better control of pulmonary pressures and to help ventilator weaning.

We report the kinetic behavior of the enzyme aldehyde oxidoreductase (AOR) from the sulfate reducing bacterium Desulfovibrio gigas (Dg) encapsulated in reverse micelles of sodium bis-(2-ethylhexyl) sulfosuccinate in isooctane using benzaldehyde, octaldehyde, and decylaldehyde as substrates. Dg AOR is a 200-kDa homodimeric protein that catalyzes the conversion of aldehydes to carboxylic acids. Ultrasedimentation analysis of Dg AOR-containing micelles showed the presence of 100-kDa molecular weight species, confirming that the Dg AOR subunits can be dissociated. UV-visible spectra of encapsulated Dg AOR are indistinguishable from the enzyme spectrum in solution, suggesting that both protein fold and metal cofactor are kept intact upon encapsulation. The catalytic constant (k(cat)) profile as a function of the micelle size W(0) (W(0)=[H(2)O]/[AOT]) using benzaldehyde as substrate showed two bell-shaped activity peaks at W(0)=20 and 26. Furthermore, enzymatic activity for octaldehyde and decylaldehyde was detected only in reverse micelles. Like for the benzaldehyde kinetics, two peaks with both similar k(cat) values and W(0) positions were obtained. EPR studies using spin-labeled reverse micelles indicated that octaldehyde and benzaldehyde are intercalated in the micelle membrane. This suggests that, though Dg AOR is found in the cytoplasm of bacterial cells, the enzyme may catalyze the reaction of substrates incorporated into a cell membrane.

The coordination features of the two dipyridine-containing polyamine macrocycles 2,5,8,11,14-pentaaza[ 15][ [15](2,2')[1,15]-bipyridylophane (L1) and 4,4'-(2,5,8,11,14-pentaaza[15]-[15](2,2')-bipyridylophane) (L2) toward Cu(II) and Ni(II) have been studied by means of potentiometric and spectrophotometric UV-vis titrations in aqueous solutions. While in L1 all the nitrogen donor atoms are convergent inside the macrocyclic cavity, in L2 the heteroaromatic nitrogen atoms are located outside. Ligands L1 and L2 form stable mono- and dinuclear complexes with Cu(II). In the case of Ni(II) coordination, only L1 gives dinuclear complexes, while L2 can form only mononuclear species. In the Cu(II) or Ni(II) complexes with L1 the metal(s) are lodged inside the macrocyclic cavity, coordinated to the heteroaromatic nitrogens. As shown by the crystal structure of the [CuL1](2+) and [NiL1](2+) cations, at least one of the two benzylic nitrogens is not coordinated and facile protonation of the complex takes place at neutral or slightly acidic pH values. The particular molecular architecture of L2, which displays two well-separated binding moieties, strongly affects its coordination behavior. In the mononuclear [ CuL2](2+) complex, the metal is encapsulated inside the cavity, not coordinated by the dipyridine unit. Protonation of the complex, however, occurs on the aliphatic polyamine chain and gives rise to translocation of the metal outside the cavity, bound to the heteroaromatic nitrogens. In the [NiL2](2+) complex the metal is coordinated by the dipyridine nitrogens, outside the macrocyclic cavity. Thermodynamic and/or kinetic considerations may explain the different behavior with respect to the corresponding Cu(II) complex.