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Pereira, AS, C. G. Timoteo, M. Guilherme, F. Folgosa, S. G. Naik, A. G. Duarte, BH HUYNH, and P. Tavares. "Spectroscopic evidence for and characterization of a trinuclear ferroxidase center in bacterial ferritin from Desulfovibrio vulgaris Hildenborough." Journal of the American Chemical Society. 134 (2012): 10822-32. AbstractWebsite

Ferritins are ubiquitous and can be found in practically all organisms that utilize Fe. They are composed of 24 subunits forming a hollow sphere with an inner cavity of ~80 A in diameter. The main function of ferritin is to oxidize the cytotoxic Fe(2+) ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe(2+) (ferroxidation) by H-type ferritins, found in vertebrates, occurs at a diiron binding center, termed the ferroxidase center. In bacterial ferritins, however, X-ray crystallographic evidence and amino acid sequence analysis revealed a trinuclear Fe binding center comprising a binuclear Fe binding center (sites A and B), homologous to the ferroxidase center of H-type ferritin, and an adjacent mononuclear Fe binding site (site C). In an effort to obtain further evidence supporting the presence of a trinuclear Fe binding center in bacterial ferritins and to gain information on the states of the iron bound to the trinuclear center, bacterial ferritin from Desulfovibrio vulgaris (DvFtn) and its E130A variant was loaded with substoichiometric amounts of Fe(2+), and the products were characterized by Mossbauer and EPR spectroscopy. Four distinct Fe species were identified: a paramagnetic diferrous species, a diamagnetic diferrous species, a mixed valence Fe(2+)Fe(3+) species, and a mononuclear Fe(2+) species. The latter three species were detected in the wild-type DvFtn, while the paramagnetic diferrous species was detected in the E130A variant. These observations can be rationally explained by the presence of a trinuclear Fe binding center, and the four Fe species can be properly assigned to the three Fe binding sites. Further, our spectroscopic data suggest that (1) the fully occupied trinuclear center supports an all ferrous state, (2) sites B and C are bridged by a mu-OH group forming a diiron subcenter within the trinuclear center, and (3) this subcenter can afford both a mixed valence Fe(2+)Fe(3+) state and a diferrous state. Mechanistic insights provided by these new findings are discussed and a minimal mechanistic scheme involving O-O bond cleavage is proposed.

Timoteo, C. G., M. Guilherme, D. Penas, F. Folgosa, P. Tavares, and AS Pereira. "Desulfovibrio vulgaris bacterioferritin uses H(2)O(2) as a co-substrate for iron oxidation and reveals DPS-like DNA protection and binding activities." The Biochemical journal. 446 (2012): 125-33. AbstractWebsite

A gene encoding Bfr (bacterioferritin) was identified and isolated from the genome of Desulfovibrio vulgaris cells, and overexpressed in Escherichia coli. In vitro, H(2)O(2) oxidizes Fe(2+) ions at much higher reaction rates than O(2). The H(2)O(2) oxidation of two Fe(2+) ions was proven by Mossbauer spectroscopy of rapid freeze-quenched samples. On the basis of the Mossbauer parameters of the intermediate species we propose that D. vulgaris Bfr follows a mineralization mechanism similar to the one reported for vertebrate H-type ferritins subunits, in which a diferrous centre at the ferroxidase site is oxidized to diferric intermediate species, that are subsequently translocated into the inner nanocavity. D. vulgaris recombinant Bfr oxidizes and stores up to 600 iron atoms per protein. This Bfr is able to bind DNA and protect it against hydroxyl radical and DNase deleterious effects. The use of H(2)O(2) as an oxidant, combined with the DNA binding and protection activities, seems to indicate a DPS (DNA-binding protein from starved cells)-like role for D. vulgaris Bfr.

Folgosa, F., C. G. Timoteo, M. Guilherme, D. Penas, P. Tavares, and AS Pereira. "Bacterioferritin from Desulfovibrio vulgaris Hildenborough is a functional DPS-like enzyme." FEBS J. 279 (2012): 465. Abstract
Timoteo, C. G., AS Pereira, C. E. Martins, S. G. Naik, A. G. Duarte, J. J. Moura, P. Tavares, BH HUYNH, and I. Moura. "Low-spin heme b(3) in the catalytic center of nitric oxide reductase from Pseudomonas nautica." Biochemistry. 50 (2011): 4251-62. AbstractWebsite

Respiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mossbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mossbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g approximately 6 and g approximately 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g approximately 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction.

Paes de Sousa, P. M., D. Rodrigues, C. G. Timoteo, M. L. Simoes Goncalves, G. W. Pettigrew, I. Moura, J. J. Moura, and M. M. Correia Dos Santos. "Analysis of the activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase through an electron transfer chain." J Biol Inorg Chem. 16 (2011): 881-8. 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.

Conrath, K., AS Pereira, C. E. Martins, C. G. Timoteo, P. Tavares, S. Spinelli, J. Kinne, C. Flaudrops, C. Cambillau, S. Muyldermans, I. Moura, J. J. Moura, M. Tegoni, and A. Desmyter. "Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductase." Protein Sci. 18 (2009): 619-28. AbstractWebsite

Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N(2)O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.

Pereira, AS, F. Folgosa, M. Guilherme, A. G. Duarte, C. G. Timóteo, P. Tavares, and BH HUYNH. "Concerted iron and oxygen detoxification at the tri-nuclear Fe site of bacterial ferritin from Desulfovibrio vulgaris Hildenborough." J Biol Inorg Chem. 14 (2009): S34. Abstract
Timoteo, C. G., C. Pantana, A. G. Duarte, F. Folgosa, AS Pereira, and P. Tavares. "The Catalytic center of a Desaturase from Arabidopsis thaliana." J Biol Inorg Chem. 12 (2007): S93. Abstract
Guilherme, M., C. G. Timoteo, P. Tavares, and AS Pereira. "Functional studies on a bacterioferritin from the anaerobe Desulfovibrio vulgaris." J Biol Inorg Chem. 12 (2007): S77. Abstract
Martins, C. E., AS Pereira, P. Tavares, C. M. Cordas, F. Folgosa, C. G. Timoteo, S. G. Naik, and BH HUYNH. "Redox states of Nitric Oxide Reductase from Pseudomonas nautica: Kinetic and Spectroscopic characterization." J Biol Inorg Chem. 12 (2007): S83. Abstract
Cordas, C. M., AS Pereira, C. E. Martins, C. G. Timoteo, I. Moura, J. J. Moura, and P. Tavares. "Nitric oxide reductase: direct electrochemistry and electrocatalytic activity." Chembiochem. 7 (2006): 1878-81. AbstractWebsite
Martins, C. E., C. M. Cordas, C. G. Timoteo, P. Tavares, and AS Pereira. "Nitric oxide reductase from Pseudomonas nautica." Eur Biophys J. 34 (2005): 663. Abstract
Timoteo, C. G., P. Tavares, C. F. Goodhew, L. C. Duarte, K. Jumel, F. M. Girio, S. Harding, G. W. Pettigrew, and I. Moura. "Ca2+ and the bacterial peroxidases: the cytochrome c peroxidase from Pseudomonas stutzeri." J Biol Inorg Chem. 8 (2003): 29-37. AbstractWebsite

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 Ca(2+) 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 Ca(2+). The affinity for Ca(2+) in the mixed valence enzyme is so high that Ca(2+) 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 Ca(2+) for formation of the dimer and also for activation of the enzyme. This is consistent with the proposal that Ca(2+) in the bacterial peroxidases influences the monomer/dimer equilibrium and the transition to the active form of the enzyme. Additional Ca(2+)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 Ca(2+)binding site of low affinity.

Bonifacio, C., CA Cunha, A. Muller, C. G. Timoteo, JM Dias, I. Moura, and MJ Romao. "Crystallization and preliminary X-ray diffraction analysis of the di-haem cytochrome c peroxidase from Pseudomonas stutzeri." Acta crystallographica. 59 (2003): 345-7. AbstractWebsite

Crystals of cytochrome c peroxidase from Pseudomonas stutzeri were obtained using sodium citrate and PEG 8000 as precipitants. A complete data set was collected to a resolution of 1.6 A under cryogenic conditions using synchrotron radiation at the ESRF. The crystals belong to space group P2(1), with unit-cell parameters a = 69.29, b = 143.31, c = 76.83 A, beta = 100.78 degrees. Four CCP molecules were found in the asymmetric unit, corresponding to a pair of dimers related by local dyads. The crystal packing in the structure shows that the functional dimers can dimerize, as suggested by previous biochemical studies.

Timoteo, C. G., P. Tavares, G. W. Pettigrew, and I. Moura. "Calcium in Bacterial Peroxidases - Pseudomonas stutzeri cytochrome c peroxidase." J Inorg Biochem. 86 (2001): 456. Abstract