Chen, P, Cabrito I, Moura JJ, Moura I, Solomon EI.
2002.
Spectroscopic and electronic structure studies of the mu(4)-sulfide bridged tetranuclear Cu(Z) cluster in N(2)O reductase: molecular insight into the catalytic mechanism, Sep 4. J Am Chem Soc. 124:10497-507., Number 35
AbstractSpectroscopic 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.
Andrade, SLA, Moura JJG.
2002.
Hydrogen evolution and consumption in AOT-isooctane reverse micelles by Desulfovibrio gigas hydrogenase, Sep 2. Enzyme and Microbial Technology. 31:398-402., Number 4
AbstractThe enzyme hydrogenase isolated from the sulphate reducing anaerobic bacterium Desulfovibrio gigas was encapsulated in reverse micelles of AOT-water-isooctane. The enzyme ability to consume molecular hydrogen was studied as a function of the micelle size (given by W-o = [H2O]/[organic solvent]). A peak of catalytic activity was obtained for W-o = 18, a micelle size theoretically fitting the heterodimeric hydrogenase molecule. At this W-o value, the recorded catalytic activity was slightly higher than in a buffer system (K-cat = 169.43 s(-1) against the buffer value of 151 s(-1)). The optimal buffer used to encapsulate the enzyme was found to be imidazole 50 mM, pH 9.0, The molecular hydrogen production activity was also tested in this reverse micelle medium. (C) 2002 Elsevier Science Inc. All lights reserved.
Raaijmakers, H, Macieira S, Dias JM, Teixeira S, Bursakov S, Huber R, Moura JJ, Moura I, Romao MJ.
2002.
Gene sequence and the 1.8 A crystal structure of the tungsten-containing formate dehydrogenase from Desulfovibrio gigas, Sep. Structure. 10:1261-72., Number 9
AbstractDesulfovibrio gigas formate dehydrogenase is the first representative of a tungsten-containing enzyme from a mesophile that has been structurally characterized. It is a heterodimer of 110 and 24 kDa subunits. The large subunit, homologous to E. coli FDH-H and to D. desulfuricans nitrate reductase, harbors the W site and one [4Fe-4S] center. No small subunit ortholog containing three [4Fe-4S] clusters has been reported. The structural homology with E. coli FDH-H shows that the essential residues (SeCys158, His159, and Arg407) at the active site are conserved. The active site is accessible via a positively charged tunnel, while product release may be facilitated, for H(+) by buried waters and protonable amino acids and for CO(2) through a hydrophobic channel.
Goodfellow, BJ, Nunes SG, Rusnak F, Moura I, Ascenso C, Moura JJ, Volkman BF, Markley JL.
2002.
Zinc-substituted Desulfovibrio gigas desulforedoxins: resolving subunit degeneracy with nonsymmetric pseudocontact shifts, Oct. Protein Sci. 11:2464-70., Number 10
AbstractDesulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S] center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with (15)N or (15)N + (13)C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D (15)N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the (13)C(alpha) shifts were obtained from an HNCA experiment. Comparison of (1)H-(15)N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues >/=8.5 A from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.
Tiago, T, Aureliano M, Duarte RO, Moura JJG.
2002.
Vanadate oligomers interaction with phosphorylated myosin, Nov 15. Inorganica Chimica Acta. 339:317-321.
AbstractUsing a myosin preparation containing endogenous myosin light-chain (LC2) kinase and phosphatase and calmodulin, i.e. near physiological ones, the interaction of vanadate oligomers with phosphorylated myosin was evaluated. Decavanadate or metavanadate solutions (2-15 mM total vanadate) did not prevent the phosphorylation state of the regulatory myosin lightchain, as observed by urea-polyacrylamide gel electrophoresis. The relative order of line broadening upon protein addition, reflecting the interaction of the vanadate oligomers with phosphorylated myosin, was V10 > V-4 > V-1 = 1 whereas, no changes were observed for monomeric vanadate. In the presence of ATP, V-1 signal was shifted upfield 2 ppm and became broadened, while V4 signal became narrowed. Moreover, a significant increase in myosin ATPase inhibition (60%) was observed when decameric vanadate species were present (1.4 mM). It is concluded that, under conditions near physiological ones, decameric vanadate differs from vanadate oligomers present in metavanadate solutions due to its strong interaction with the phosphorylated enzyme and myosin ATPase inhibition. Besides, ATP decreases the affinity of myosin for tetravanadate, induces the interaction with monomeric vanadate, whereas it does not affect decameric vanadate interaction. (C) 2002 Elsevier Science B.V. All rights reserved.
Sola, S, Brito MA, Brites D, Moura JJG, Rodrigues CMP.
2002.
Membrane structural changes support the involvement of mitochondria in the bile salt-induced apoptosis of rat hepatocytes, Nov. Clinical Science. 103:475-485., Number 5
AbstractThe accumulation of toxic bile salts within the hepatocyte plays a key role in organ injury during liver disease. Deoxycholate (DC) and glycochenodeoxycholate (GCDC) induce apoptosis in vitro and in vivo, perhaps through direct perturbation of mitochondrial membrane structure and function. In contrast, ursodeoxycholate (UDC) and its taurine-conjugated form (TUDC) appear to be protective. We show here that hydrophobic bile salts induced apoptosis in cultured rat hepatocytes, without modulating the expression of pro-apoptotic Bax protein, and caused cytochrome c release in isolated mitochondria. Co-incubation with UDC and TUDC prevented cell death and efflux of mitochondrial factors. Using spin-labelling techniques and EPR spectroscopy analysis of isolated rat liver mitochondria, we found significant structural changes at the membrane-water surface in mitochondria exposed to hydrophobic bile salts, including modified lipid polarity and fluidity, altered protein order and increased oxidative injury. UDC, TUDC and cyclosporin A almost completely abrogated DC- and GCDC-induced membrane perturbations. We conclude that the toxicity of hydrophobic bile salts to hepatocytes is mediated by cytochrome c release, through a mechanism associated with marked direct effects on mitochondrial membrane lipid polarity and fluidity, protein order and redox status, without modulation of pro-apoptotic Bax expression. UDC and TUDC can directly suppress disruption of mitochondrial membrane structure, which may represent an important mechanism of hepatoprotection by these bile salts.
Moniz, AB.
2002.
A contribui{\c c}ão da Sociologia para a forma{\c c}ão em Engenharia[Contribution of sociology to the engineering training], May. , Number 8103: University Library of Munich, Germany
AbstractThis article is based on the lesson presented in the scope of academy activities in the area of Sociology (at FCT-UNL). It is intended to approach the controversies concerning the relation between technology and society (technological determinism, effect on employment, importance of the social behaviours in the definition of needs for new products and equipment), and on the most recent trends (over all, since middle of the last century) in terms of technological evolution and of its social and cultural change. Finally, this subject was dedicated to the presentation of the main factors that has lead to the development of the contribution of sociology for the training and education in engineering. Thus, one intends to acquire new elements on this area of knowledge also presented in other schools of engineering of other countries (for examples, United States and Holland), and how the theoretical beddings are been developed since the beginning of 20th century on the modalities of work organization that imply a cooperation between engineering and social sciences.
Moniz, AB.
2002.
{A contribuição da Sociologia para a formação em Engenharia[Contribution of sociology to the engineering training]}, May. , Number 8103: University Library of Munich, Germany
AbstractThis article is based on the lesson presented in the scope of academy activities in the area of Sociology (at FCT-UNL). It is intended to approach the controversies concerning the relation between technology and society (technological determinism, effect on employment, importance of the social behaviours in the definition of needs for new products and equipment), and on the most recent trends (over all, since middle of the last century) in terms of technological evolution and of its social and cultural change. Finally, this subject was dedicated to the presentation of the main factors that has lead to the development of the contribution of sociology for the training and education in engineering. Thus, one intends to acquire new elements on this area of knowledge also presented in other schools of engineering of other countries (for examples, United States and Holland), and how the theoretical beddings are been developed since the beginning of 20th century on the modalities of work organization that imply a cooperation between engineering and social sciences.
Rodrigues, CM, Sola S, Brito MA, Brites D, Moura JJ.
2002.
Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria, Mar. J Hepatol. 36:335-41., Number 3
AbstractBACKGROUND/AIMS: Unconjugated bilirubin (UCB) impairs crucial aspects of cell function and induces apoptosis in primary cultured neurones. While mechanisms of cytotoxicity begin to unfold, mitochondria appear as potential primary targets. METHODS: We used electron paramagnetic resonance spectroscopy analysis of isolated rat mitochondria to test the hypothesis that UCB physically interacts with mitochondria to induce structural membrane perturbation, leading to increased permeability, and subsequent release of apoptotic factors. RESULTS: Our data demonstrate profound changes on mitochondrial membrane properties during incubation with UCB, including modified membrane lipid polarity and fluidity (P<0.01), as well as disrupted protein mobility (P<0.001). Consistent with increased permeability, cytochrome c was released from the intermembrane space (P<0.01), perhaps uncoupling the respiratory chain and further increasing oxidative stress (P<0.01). Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated UCB-induced perturbation. CONCLUSIONS: UCB directly interacts with mitochondria influencing membrane lipid and protein properties, redox status, and cytochrome c content. Thus, apoptosis induced by UCB may be mediated, at least in part, by physical perturbation of the mitochondrial membrane. These novel findings should ultimately prove useful to our evolving understanding of UCB cytotoxicity.