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)
AbstractA 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.
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)
AbstractA 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.
Biochemical and spectroscopic characterization of overexpressed fuscoredoxin from Escherichia coli,
Pereira, A. S., Tavares P., Krebs C., Huynh B. H., Rusnak F., Moura I., and Moura J. J.
, Biochem Biophys Res Commun, Jun 24, Volume 260, Number 1, p.209-15, (1999)
AbstractFuscoredoxin is a unique iron containing protein of yet unknown function originally discovered in the sulfate reducers of the genus Desulfovibrio. It contains two iron-sulfur clusters: a cubane [4Fe-4S] and a mixed oxo- and sulfido-bridged 4Fe cluster of unprecedented structure. The recent determination of the genomic sequence of Escherichia coli (E. coli) has revealed a homologue of fuscoredoxin in this facultative microbe. The presence of this gene in E. coli raises interesting questions regarding the function of fuscoredoxin and whether this gene represents a structural homologue of the better-characterized Desulfovibrio proteins. In order to explore the latter, an overexpression system for the E. coli fuscoredoxin gene was devised. The gene was cloned from genomic DNA by use of the polymerase chain reaction into the expression vector pT7-7 and overexpressed in E. coli BL21(DE3) cells. After two chromatographic steps a good yield of recombinant protein was obtained (approximately 4 mg of pure protein per liter of culture). The purified protein exhibits an optical spectrum characteristic of the homologue from D. desulfuricans, indicating that cofactor assembly was accomplished. Iron analysis indicated that the protein contains circa 8 iron atoms/molecule which were shown by EPR and Mossbauer spectroscopies to be present as two multinuclear clusters, albeit with slightly altered spectroscopic features. A comparison of the primary sequences of fuscoredoxins is presented and differences on cluster coordination modes are discussed on the light of the spectroscopic data.
Biochemical characterization of the purple form of Marinobacter hydrocarbonoclasticus nitrous oxide reductase,
Dell'Acqua, S., Pauleta S. R., Moura J. J., and Moura I.
, Philos Trans R Soc Lond B Biol Sci, Volume 367, Issue 1593, p.1204-1212, (2012)
Biosensor for direct bioelectrocatalysis detection of nitric oxide using nitric oxide reductase incorporated in carboxylated single-walled carbon nanotubes/lipidic bilayer nanocomposite,
Gomes, FO, Maia L. B., Loureiro JA, Pereira MC, Delerue-Matos C., Moura I., Moura J. J. G., and Morais S.
, Bioelectrochem, Volume 127, p.76-86, (2019)
A bird’s-eye view of denitrification in relation to the nitrogen cycle,
Moura, I., Maia L. B., Pauleta S. R., and Moura J. J. G.
, Metalloenzymes in Denitrification: Applications and Environmental Impacts, RSC Metallobiology Series No. 9 (ISBN: 978-1-78262-376-2)., Cambridge, p.1-10, (2017)