Publications

Export 528 results:
Sort by: [ Author  (Asc)] Title Type Year
A B C D E F G H I J K L M N O P Q R S T U V W X Y Z 
N
Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum, Najmudin, S., Gonzalez P. J., Trincao J., Coelho C., Mukhopadhyay A., Cerqueira N. M., Romao C. C., Moura I., Moura J. J., Brondino C. D., and Romao M. J. , J Biol Inorg Chem, Jun, Volume 13, Number 5, p.737-53, (2008) AbstractWebsite

Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 A. The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH(2) ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (lambda = 1.77 A) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sgamma atom of the molybdenum ligand Cys(A140) was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 A, indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with (15)N-labeled nitrate and in D(2)O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)-nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.

ArsC3 from Desulfovibrio alaskensis G20, a cation and sulfate-independent highly efficient arsenate reductase, Nunes, C. I., Brás J. L., Najmudin S., Moura J. J. G., Moura I., and Carepo M. , J Biol Inorg Chem, Volume 19, p.1277-1285, (2014)
Evaluation of Sweat Sampling Procedures for Human Stress Biomarkers Detection, Nunes, M. J., Moura J. J. G., Noronha J. P., Branco L. C., Samhan-Arias A., Sousa J. P., Rouco C., and Cordas C. , Analytica, Volume 3, p.178–194, (2022)
Screening of Potential Stress Biomarkers in Sweat Associated with Sports Training, Nunes, M. J., Cordas C. M., Moura J. J. G., Noronha J. P., and Branco L. C. , Sports Medicine - Open, Volume 7, p.8, (2021)
O
Study of membrane ageing and grafting mechanisms using electron paramagnetic resonance, Oliveira, F. R. P., Matos C. T., Moura J. J. G., Portugal C. A. M., and Crespo J. G. , Desalination Water Treatment, Volume 27, p.141–149, (2011)
P
Insights into the electrochemical behaviour of composite materials: Monovacant polyoxometalates porous metal-organic framework, Paes de Sousa, P. M., Grazina R., Barbosa A. D. S., de Castro Baltazar, Moura J. J. G., Cunha-Silva L., and Salete S. , Electrochim Acta, Volume 87, p.853-859, (2013)
Analysis of the activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase through an electron transfer chain, Paes de Sousa, P. M., Rodrigues D., Timoteo C. G., Simoes Goncalves M. L., Pettigrew G. W., Moura I., Moura J. J., and Correia dos Santos M. M. , J Biol Inorg Chem, Aug, Volume 16, Number 6, p.881-8, (2011) AbstractWebsite

The activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase (CCP) was probed through the mediated electrochemical catalysis by its physiological electron donor, P. stutzeri cytochrome c-551. A comparative study was carried out, by performing assays with the enzyme in the resting oxidized state as well as in the mixed-valence activated form, using cyclic voltammetry and a pyrolytic graphite membrane electrode. In the presence of both the enzyme and hydrogen peroxide, the peak-like signal of cytochrome c-551 is converted into a sigmoidal wave form characteristic of an E(r)C'(i) catalytic mechanism. An intermolecular electron transfer rate constant of (4 +/- 1) x 10(5) M(-1) s(-1) was estimated for both forms of the enzyme, as well as a similar Michaelis-Menten constant. These results show that neither the intermolecular electron transfer nor the catalytic activity is kinetically controlled by the activation mechanism of CCP in the case of the P. stutzeri enzyme. Direct enzyme catalysis using protein film voltammetry was unsuccessful for the analysis of the activation mechanism, since P. stutzeri CCP undergoes an undesirable interaction with the pyrolytic graphite surface. This interaction, previously reported for the Paracoccus pantotrophus CCP, induces the formation of a non-native conformation state of the electron-transferring haem, which has a redox potential 200 mV lower than that of the native state and maintains peroxidatic activity.

Benefits of membrane electrodes in the electrochemistry of metalloproteins: mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by horse cytochrome c: a case study, Paes de Sousa, P. M., Pauleta S. R., Rodrigues D., Simoes Goncalves M. L., Pettigrew G. W., Moura I., Moura J. J., and Correia dos Santos M. M. , J Biol Inorg Chem, Jun, Volume 13, Number 5, p.779-87, (2008) AbstractWebsite

A comparative study of direct and mediated electrochemistry of metalloproteins in bulk and membrane-entrapped solutions is presented. This work reports the first electrochemical study of the electron transfer between a bacterial cytochrome c peroxidase and horse heart cytochrome c. The mediated catalysis of the peroxidase was analysed both using the membrane electrode configuration and with all proteins in solution. An apparent Michaelis constant of 66 +/- 4 and 42 +/- 5 microM was determined at pH 7.0 and 0 M NaCl for membrane and bulk solutions, respectively. The data revealed that maximum activity occurs at 50 mM NaCl, pH 7.0, with intermolecular rate constants of (4.4 +/- 0.5) x 10(6) and (1.0 +/- 0.5) x 10(6) M(-1) s(-1) for membrane-entrapped and bulk solutions, respectively. The influence of parameters such as pH or ionic strength on the mediated catalytic activity was analysed using this approach, drawing attention to the fact that careful analysis of the results is needed to ensure that no artefacts are introduced by the use of the membrane configuration and/or promoters, and therefore the dependence truly reflects the influence of these parameters on the (mediated) catalysis. From the pH dependence, a pK of 7.5 was estimated for the mediated enzymatic catalysis.

Artefacts induced on c-type haem proteins by electrode surfaces, Paes de Sousa, P. M., Pauleta S. R., Simoes Goncalves M. L., Pettigrew G. W., Moura I., Moura J. J., and Correia dos Santos M. M. , J Biol Inorg Chem, Feb, Volume 16, Number 2, p.209-15, (2011) AbstractWebsite

In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met-His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c(3) from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.

Synechocystis ferredoxin/ferredoxin-NADP(+)-reductase/NADP+ complex: Structural model obtained by NMR-restrained docking, Palma, P. N., Lagoutte B., Krippahl L., Moura J. J., and Guerlesquin F. , FEBS Lett, Aug 29, Volume 579, Number 21, p.4585-90, (2005) AbstractWebsite

Ferredoxin (Fd) and ferredoxin-NADP(+)-reductase (FNR) are two terminal physiological partners of the photosynthetic electron transport chain. Based on a nuclear magnetic resonance (NMR)-restrained-docking approach, two alternative structural models of the Fd-FNR complex in the presence of NADP+ are proposed. The protein docking simulations were performed with the software BiGGER. NMR titration revealed a 1:1 stoichiometry for the complex and allowed the mapping of the interacting residues at the surface of Fd. The NMR chemical shifts were encoded into distance constraints and used with theoretically calculated electronic coupling between the redox cofactors to propose experimentally validated docked complexes.

BiGGER: a new (soft) docking algorithm for predicting protein interactions, Palma, P. N., Krippahl L., Wampler J. E., and Moura J. J. , Proteins, Jun 1, Volume 39, Number 4, p.372-84, (2000) AbstractWebsite

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.

Evidence for a ternary complex formed between flavodoxin and cytochrome c3: 1H-NMR and molecular modeling studies, Palma, P. N., Moura I., Legall J., Van Beeumen J., Wampler J. E., and Moura J. J. , Biochemistry, May 31, Volume 33, Number 21, p.6394-407, (1994) AbstractWebsite

Small electron-transfer proteins such as flavodoxin (16 kDa) and the tetraheme cytochrome c3 (13 kDa) have been used to mimic, in vitro, part of the complex electron-transfer chain operating between substrate electron donors and respiratory electron acceptors, in sulfate-reducing bacteria (Desulfovibrio species). The nature and properties of the complex formed between these proteins are revealed by 1H-NMR and molecular modeling approaches. Our previous study with the Desulfovibrio vulgaris proteins [Moura, I., Moura, J.J. G., Santos, M.H., & Xavier, A. V. (1980) Cienc. Biol. (Portugal) 5, 195-197; Stewart, D.E. LeGall, J., Moura, I., Moura, J. J. G., Peck, H.D. Jr., Xavier, A. V., Weiner, P. K., & Wampler, J.E. (1988) Biochemistry 27, 2444-2450] indicated that the complex between cytochrome c3 and flavodoxin could be monitored by changes in the NMR signals of the heme methyl groups of the cytochrome and that the electrostatic surface charge (Coulomb's law) on the two proteins favored interaction between one unique heme of the cytochrome with flavodoxin. If the interaction is indeed driven by the electrostatic complementarity between the acidic flavodoxin and a unique positive region of the cytochrome c3, other homologous proteins from these two families of proteins might be expected to interact similarly. In this study, three homologous Desulfovibrio cytochromes c3 were used, which show a remarkable variation in their individual isoelectric points (ranging from 5.5 to 9.5). On the basis of data obtained from protein-protein titrations followed at specific proton NMR signals (i.e., heme methyl resonances), a binding model for this complex has been developed with evaluation of stoichiometry and binding constants. This binding model involves one site on the cytochromes c3 and two sites on the flavodoxin, with formation of a ternary complex at saturation. In order to understand the potential chemical form of the binding model, a structural model for the hypothetical ternary complex, formed between one molecule of Desulfovibrio salexigens flavodoxin and two molecules of cytochrome c3, is proposed. These molecular models of the complexes were constructed on the basis of complementarity of Coulombic electrostatic surface potentials, using the available X-ray structures of the isolated proteins and, when required, model structures (D. salexigens flavodoxin and Desulfovibrio desulfuricans ATCC 27774 cytochrome c3) predicted by homology modeling.

Moessbauer study of D. gigas ferredoxin II and spin-coupling model for Fe3S4 cluster with valence delocalization, Papaefthymiou, V., Girerd J. J., Moura I., Moura J. J. G., and Muenck E. , Journal of the American Chemical Society, 1987/07/01, Volume 109, Number 15, p.4703-4710, (1987) AbstractWebsite
n/a
EPR-detectable redox centers of the periplasmic hydrogenase from Desulfovibrio vulgaris, Patil, D. S., Moura J. J., He S. H., Teixeira M., Prickril B. C., Dervartanian D. V., Peck, H. D. Jr., Legall J., and Huynh B. H. , J Biol Chem, Dec 15, Volume 263, Number 35, p.18732-8, (1988) AbstractWebsite

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) belongs to the category of [Fe] hydrogenase which contains only iron-sulfur clusters as its prosthetic groups. Amino acid analyses were performed on the purified D. vulgaris hydrogenase. The amino acid composition obtained compared very well with the result derived from the nucleotide sequence of the structural gene (Voordouw, G., Brenner, S. (1985) Eur. J. Biochem. 148, 515-520). Detailed EPR reductive titration studies on the D. vulgaris hydrogenase were performed to characterize the metal centers in this hydrogenase. In addition to the three previously observed EPR signals (namely, the "isotropic" 2.02 signal, the rhombic 2.10 signal, and the complex signal of the reduced enzyme), a rhombic signal with resonances at the g-values of 2.06, 1.96, and 1.89 (the rhombic 2.06 signal) was detected when the samples were poised at potentials between 0 and -250 mV (with respect to normal hydrogen electrode). The midpoint redox potentials for each of the four EPR-active species were determined, and the characteristics of each EPR signal are described. Both the rhombic 2.10 and 2.06 signals exhibit spectral properties that are distinct from a ferredoxin-type [4Fe-4S] cluster and are proposed to originate from the same H2-binding center but in two different conformations. The complex signal of the reduced hydrogenase has been shown to represent two spin-spin interacting ferredoxin-type [4Fe-4S]1+ clusters (Grande, H. J., Dunham, W. R., Averill, B., Van Dijk, C., and Sands, R. H. (1983) Eur. J. Biochem. 136, 201-207). The titration data indicated a strong cooperative effect between these two clusters during their reduction. In an effort to accurately estimate the number of iron atoms/molecule of hydrogenase, plasma emission and chemical methods were used to determine the iron contents in the samples; and four different methods, including amino acid analysis, were used for protein determination. The resulting iron stoichiometries were found to be method-dependent and vary over a wide range (+/- 20%). The uncertainties involved in the determination of iron stoichiometry are discussed.

NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo-Cu cluster, Pauleta, S. R., Duarte A. G., Carepo M. S., Pereira A. S., Tavares P., Moura I., and Moura J. J. , Biomol NMR Assign, Jul, Volume 1, Number 1, p.81-3, (2007) AbstractWebsite

We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo-Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using 13C-detection experiments.

Source and reduction of nitrous oxide, Pauleta, S. R., Carepo M. S., and Moura I. , Coord Chem Rev, Volume 387, p.436-449, (2019)
Iron-Sulfur centers: Functions of an ancient metal site, Pauleta, S. R., Carepo M., Grazina R., Moura I., and Moura J. J. G. , Comprehensive Inorganic Chemistry III From Biology to Nanotechnology, vol. 2, ???, p.???, (2022)
Marinobacter hydrocarbonoclasticus is an aerobic denitrifier, Pauleta, S. R., Ramos S., Pietsch M., Carreira C., Dell'Acqua S., and Moura I. , EuroBIC 11, Granada, p.49-53, (2013)
Insights into nitrous oxide reductase, Pauleta, S. R., Carreira C., and Moura I. , Metalloenzymes in Denitrification: Applications and Environmental Impacts, RSC Metallobiology Series No. 9 (ISBN: 978-1-78262-376-2)., p.141-169, (2017)
Nitrous Oxide Reductase, Pauleta, S. R., Dell’Acqua S., and Moura I. , Coord Chem Rev, Volume 257, p.332-249, (2013)
The treta copper-sulfide center of nitrous oxide reductase, Pauleta, S. R., Carepo M. S., and Moura I. , Transition Metals and Sulfur- A Strong Relationship with Life, Berlin, p.Chapter 5, (2019)
Paracoccus pantotrophus pseudoazurin is an electron donor to cytochrome c peroxidase, Pauleta, S. R., Guerlesquin F., Goodhew C. F., Devreese B., Van Beeumen J., Pereira A. S., Moura I., and Pettigrew G. W. , Biochemistry, Sep 7, Volume 43, Number 35, p.11214-11225, (2004) AbstractWebsite

The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was re-evaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic- strength-dependent. The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.

A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase, Pauleta, S. R., Cooper A., Nutley M., Errington N., Harding S., Guerlesquin F., Goodhew C. F., Moura I., Moura J. J., and Pettigrew G. W. , Biochemistry, Nov 23, Volume 43, Number 46, p.14566-76, (2004) AbstractWebsite

Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K(d) of 16.4 microM at 25 degrees C, pH 6.0, in an endothermic reaction that is driven by a large entropy change. Sedimentation velocity experiments confirmed the presence of a single site, although results at higher pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry, ultracentrifugation, and (1)H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm. Molecular docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were situated close to the electron-transferring heme with Cu-Fe distances of about 14 A. This is consistent with the results of (1)H NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electron-transferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme.

Calcium-dependent conformation of a heme and fingerprint peptide of the diheme cytochrome c peroxidase from Paracoccus pantotrophus, Pauleta, S. R., Lu Y., Goodhew C. F., Moura I., Pettigrew G. W., and Shelnutt J. A. , Biochemistry, Jun 5, Volume 40, Number 22, p.6570-6579, (2001) AbstractWebsite

The structural changes in the heme macrocycle and substituents caused by binding of Ca2+ to the diheme cytochrome c peroxidase from Paracoccus pantotrophus were clarified by resonance Raman spectroscopy of the inactive fully oxidized form of the enzyme. The changes in the macrocycle vibrational modes are consistent with a Ca2+-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme. Most of the increase in out-of-plane distortion occurs when the high-affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca2+ or Mg2+. This increase in the heme distortion explains the red shift in the Soret absorption band that occurs upon Ca2+ binding. Changes also occur in the low-frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon Ca2+ binding to site I. These structural changes may lead to loss of the sixth ligand at the peroxidatic heme in the semireduced form of the enzyme and activation.

Electron transfer and docking between cytochrome cd1 nitrite reductase and different redox partners - A comparative study, Pedroso, H. A., Silveira C. M., Almeida R. M., Almeida A., Besson S., Moura I., Moura J. J. G., and Almeida M. G. , Biochim Biophys Acta, Volume 1857, p.1412-142104.279, (2016) Website