Antonio J. Costa-Filho

Mono- and polynuclear complexes of Fe(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) with N,N′-bis(3-hydroxysalicylidene)-1,3-diamino-2-propanol

Abstract Mono- and polynuclear neutral complexes have been obtained by direct electrochemical reaction of metal anodes with the potentially heptadentate and tetracompartmental ligand N , N ′-bis(3-hydroxysalicylidene)-1,3-diamino-2-propanol (H 5 L). Physicochemical data suggest that the ligand behaves as a di- or tetra-anion in mono- and polynuclear complexes, respectively. Metal ions are held together by μ-phenoxo oxygen bridges in polynuclear complexes. Nickel and copper environments are distorted to tetrahedral and square-planar, respectively. Zinc and cadmium could be hexa-coordinated in the mononuclear complexes. Finally, iron and cobalt ions seem to be in pseudo-octahedral fields.

Engineering Bifunctional Laccase-Xylanase Chimeras for Improved Catalytic Performance

Background: Rational design methods can be used to create chimeric enzymes with novel catalytic combinations. Results: Bifunctional enzymes combining xylanase and laccase activities showed enhanced catalytic activity and stability. Conclusion: Formation of an inter-domain interface alters enzyme conformation that enhances catalytic performance of the chimera. Significance: Deeper understanding of structural principles of protein fusion can improve the design of novel catalysts. Two bifunctional enzymes exhibiting combined xylanase and laccase activities were designed, constructed, and characterized by biochemical and biophysical methods. The Bacillus subtilis cotA and xynA genes were used as templates for gene fusion, and the xynA coding sequence was inserted into a surface loop of the cotA. A second chimera was built replacing the wild-type xynA gene by a thermostable variant (xynAG3) previously obtained by in vitro molecular evolution. Kinetic measurements demonstrated that the pH and temperature optima of the catalytic domains in the chimeras were altered by less than 0.5 pH units and 5 °C, respectively, when compared with the parental enzymes. In contrast, the catalytic efficiency (kcat/Km) of the laccase activity in both chimeras was 2-fold higher than for the parental laccase. Molecular dynamics simulations of the CotA-XynA chimera indicated that the two domains are in close contact, which was confirmed by the low resolution structure obtained by small angle x-ray scattering. The simulation also indicates that the formation of the inter-domain interface causes the dislocation of the loop comprising residues Leu-558 to Lys-573 in the laccase domain, resulting in a more accessible active site and exposing the type I Cu2+ ion to the solvent. These structural changes are consistent with the results from UV-visible electronic and EPR spectroscopy experiments of the type I copper between the native and chimeric enzymes and are likely to contribute to the observed increase in catalytic turnover number.

Effects of the antimalarial drug primaquine on the dynamic structure of lipid model membranes.

Primaquine (PQ) is a potent therapeutic agent used in the treatment of malaria and its mechanism of action still lacks a more detailed understanding at a molecular level. In this context, we used differential scanning calorimetry (DSC), pressure perturbation calorimetry (PPC), and electron spin resonance (ESR) to investigate the effects of PQ on the lipid phase transition, acyl chain dynamics, and on volumetric properties of lipid model membranes. DSC thermograms revealed that PQ stabilizes the fluid phase of the lipid model membranes and interacts mainly with the lipid headgroups. This result was revealed by the great effect on the pretransition of phosphatidylcholines and the destabilization of the inverted hexagonal phase of a phosphatidylethanolamine bilayer. Spin probes located at different positions along the lipid chain were used to monitor different membrane regions. ESR results indicated that PQ is effective in changing the acyl chain ordering and dynamics of the whole chain of dimyristoylphosphatidylcholine (DMPC) phospholipid in the rippled gel phase. The combined ESR and PPC results revealed that the slight DMPC volume changes at the main phase transition induced by the presence of PQ is probably due to a less dense lipid gel phase. At physiological pH, the cationic amphiphilic PQ strongly interacts with the lipid headgroup region of the bilayers, causing considerable disorganization in the hydrophobic core. These results shed light on the molecular mechanism of primaquine-lipid interaction, which may be useful in the understanding of the complex mechanism of action and/or the adverse effects of this antimalarial drug.

TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation

Orally administered nitrite exerts antihypertensive effects associated with increased gastric nitric oxide (NO) formation. While reducing agents facilitate NO formation from nitrite, no previous study has examined whether antioxidants with reducing properties improve the antihypertensive responses to orally administered nitrite. We hypothesized that TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) could enhance the hypotensive effects of nitrite in hypertensive rats by exerting antioxidant effects (and enhancing NO bioavailability) and by promoting gastric nitrite-derived NO generation. The hypotensive effects of intravenous and oral sodium nitrite were assessed in unanesthetized freely moving rats with L-NAME (N(ω)-nitro-L-arginine methyl ester; 100mg/kg; po)-induced hypertension treated with TEMPOL (18mg/kg; po) or vehicle. While TEMPOL exerted antioxidant effects in hypertensive rats, as revealed by lower plasma 8-isoprostane and vascular reactive oxygen species levels, this antioxidant did not affect the hypotensive responses to intravenous nitrite. Conversely, TEMPOL enhanced the dose-dependent hypotensive responses to orally administered nitrite, and this effect was associated with higher increases in plasma nitrite and lower increases in plasma nitrate concentrations. In vitro experiments using electrochemical and chemiluminescence NO detection under variable pH conditions showed that TEMPOL enhanced nitrite-derived NO formation, especially at low pH (2.0 to 4.0). TEMPOL signal evaluated by electron paramagnetic resonance decreased when nitrite was reduced to NO under acidic conditions. Consistent with these findings, increasing gastric pH with omeprazole (30mg/kg; po) attenuated the hypotensive responses to nitrite and blunted the enhancement in plasma nitrite concentrations and hypotensive effects induced by TEMPOL. Nitrite-derived NO formation in vivo was confirmed by using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), which blunted the responses to oral nitrite. Our results showed that TEMPOL promotes nitrite reduction to NO in the stomach and enhanced plasma nitrite concentrations and the hypotensive effects of oral sodium nitrite through mechanisms critically dependent on gastric pH. Interestingly, the effects of TEMPOL on nitrite-mediated hypotension cannot be explained by increased NO formation in the stomach alone, but rather appear more directly related to increased plasma nitrite levels and reduced nitrate levels during TEMPOL treatment. This may relate to enhanced nitrite uptake or reduced nitrate formation from NO or nitrite.

Crystal structure and exchange pathways of the complex l-(trypthophyl-glycinato)copper(II)

Abstract The crystal structure and the EPR characterization of the compound Cu [C13H13N3O3] is reported. It crystallizes in the P212121 space group, with a=8.2829(5), b=9.347(2), c=16.499(2) A and Z=4. The copper ion is in a distorted square planar coordination, bonded to two nitrogen and one oxygen atoms from one dipeptide and to an oxygen atom from a symmetry-related molecule. Thus, neighbor copper atoms at 5.14 A are connected by equatorial syn–anti carboxylate bridges giving rise to a chain structure along the b-axis. The chains are connected via hydrogen bonds and cation–π interactions, the latter being provided by the ‘sandwich’ structure involving each copper atom and two tryptophan residues from neighbor molecules. The EPR spectra of polycrystalline sample imply an essentially dx2−y2 ground state orbital for the Cu(II) ions. The g-values reflect a slightly distorted axial symmetry around the Cu(II) ions as expected from the structural results. No hyperfine interaction is observed, which is indicative of the presence of exchange interactions between the copper atoms as suggested by the X-ray results as well.

Cu(II) complexation with His-Gly and His-Ala. X-ray structure of [Cu(his-gly)2(H2O)2].6H2O

Abstract Two novel Cu(II)–dipeptide complexes containing l -histidyl– l -glycine and l -histidyl– l -alanine as ligands were prepared and characterised by infrared and electronic spectroscopies. EPR measurements were also made in solution and in the solid state. Cyclic voltammetric studies were carried out on aqueous solution. The crystalline structure of the [Cu( l -histidyl– l -glycine) 2 (H 2 O) 2 ]·6H 2 O was determined by X-ray diffractometry. It crystallises in the space group C 2. The Cu(II) is in a tetragonally distorted octahedral coordination, where the basal plane is occupied by N-atoms belonging to the histidine portion. The apical positions are occupied by two molecules of water.

Synthesis, structural characterization and cytotoxic activity of ternary copper(II)–dipeptide–phenanthroline complexes. A step towards the development of new copper compounds for the treatment of cancer

In the search for new compounds with antitumor activity, coordination complexes with different metals are being studied by our group. This work presents the synthesis and characterization of six copper complexes with general stoichiometry [Cu(L-dipeptide)(phen)]·nH2O (were phen=1,10-phenanthroline) and their cytotoxic activities against tumor cell lines. To characterize these systems, analytical and spectroscopic studies were performed in solid state (by UV-visible, IR, X-ray diffraction) including the crystal structure of four new complexes (of the six complexes studied): [Cu(Ala-Phe)(phen)]·4H2O, [Cu(Phe-Ala)(phen)]·4H2O, [Cu(Phe-Val)(phen)]·4.5H2O and [Cu(Phe-Phe)(phen)]·3H2O. In all of them, the copper ion is situated in a distorted squared pyramidal environment. The phen ligand is perpendicular to the dipeptide, therefore exposed and potentially available for interaction with biological molecules. In addition, for all the studied complexes, structural information in solution using EPR and UV-visible spectroscopies were obtained, showing that the coordination observed in solid state is maintained. The lipophilicity, DNA binding and albumin interaction were also studied. Biological experiments showed that all the complexes induce cell death in the cell lines: HeLa (human cervical adenocarcinoma), MCF-7 (human metastatic breast adenocarcinoma) and A549 (human lung epithelial carcinoma). Among the six complexes, [Cu(Ala-Phe)(phen)] presents the lowest IC50 values. Taken together all these data we hypothesize that [Cu(Ala-Phe)(phen)] may be a good candidate for further studies in vivo.

EPR and electrochemistry of [NH4]trans-[RuCl4(DMSO)(L)] complexes (L = DMSO, py ). X-ray molecular structure of [pyH][RuCl4(DMSO)(py)]

O complexo [(DMSO)2H]trans-[RuCl4 (DMSO)2], com simetria axial, apresenta em seu espectro de RPE dois valores de g (g^=2,35 e g//=1,87). O complexo [NH4]trans-[RuCl4(DMSO) 2] apresenta somente um valor de g no espectro RPE no estado solido, indicando acoplamento entre os microcristais. Em solucao recem-preparada, utilizando-se MeOH, detectam-se por RPE os compostos [NH4]trans-[RuCl4(DMSO)(MeOH)] e o [RuCl3(DMSO)(MeOH)2]. Apos aproximadamente 15 h apenas este ultimo composto encontra-se em solucao. Estas especies sao detectadas por voltametria ciclica. A especie [RuCl2(DMSO)(MeOH)3] e gerada eletroquimicamente a partir do [RuCl3(DMSO)(MeOH)2]. O complexo [pyH]trans-[RuCl4(DMSO)(py)], obtido a partir da reacao entre o [(DMSO)2H]trans-[RuCl4 (DMSO)2] e py, cristalizou no grupo espacial P (No.2), Z=2 com a=7,7608(1), b=8,5451(1), c=15,095(5)A, b=79,33(2)o. A estrutura foi resolvida pelas tecnicas de Patterson e diferenciais de Fourier e refinadas para R = 0,0886. O espectro de RPE (T = -160 oC) confirmou a presenca de Ru(III) paramagneticamente ativo e e consistente com uma simetria axial para o complexo (g^=2,42 e g//=1,81). O voltamograma ciclico do complexo com a piridina apresenta E1/2= - 0,15 V (vs. Ag/AgCl).

Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzymes N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzymes active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

Dynamics and Conformational Studies of TOAC Spin Labeled Analogues of Ctx(Ile21)-Ha Peptide from Hypsiboas albopunctatus

Antimicrobial peptides (AMPs) isolated from several organisms have been receiving much attention due to some specific features that allow them to interact with, bind to, and disrupt cell membranes. The aim of this paper was to study the interactions between a membrane mimetic and the cationic AMP Ctx(Ile21)-Ha as well as analogues containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) incorporated at residue positions n = 0, 2, and 13. Circular dichroism studies showed that the peptides, except for [TOAC13]Ctx(Ile21)-Ha, are unstructured in aqueous solution but acquire different amounts of α-helical secondary structure in the presence of trifluorethanol and lysophosphocholine micelles. Fluorescence experiments indicated that all peptides were able to interact with LPC micelles. In addition, Ctx(Ile21)-Ha and [TOAC13]Ctx(Ile21)-Ha peptides presented similar water accessibility for the Trp residue located near the N-terminal sequence. Electron spin resonance experiments showed two spectral components for [TOAC0]Ctx(Ile21)-Ha, which are most likely due to two membrane-bound peptide conformations. In contrast, TOAC2 and TOAC13 derivatives presented a single spectral component corresponding to a strong immobilization of the probe. Thus, our findings allowed the description of the peptide topology in the membrane mimetic, where the N-terminal region is in dynamic equilibrium between an ordered, membrane-bound conformation and a disordered, mobile conformation; position 2 is most likely situated in the lipid polar head group region, and residue 13 is fully inserted into the hydrophobic core of the membrane.