RESUMO
Natural brown-black eumelanin pigments confer structural coloration in animals and potently block ionizing radiation and antifungal drugs. These functions also make them attractive for bioinspired materials design, including coating materials for drug-delivery vehicles, strengthening agents for adhesive hydrogel materials, and free-radical scavengers for soil remediation. Nonetheless, the molecular determinants of the melanin "developmental road traveled" and the resulting architectural features have remained uncertain because of the insoluble, heterogeneous, and amorphous characteristics of these complex polymeric assemblies. Here, we used 2D solid-state NMR, EPR, and dynamic nuclear polarization spectroscopic techniques, assisted in some instances by the use of isotopically enriched precursors, to address several open questions regarding the molecular structures and associated functions of eumelanin. Our findings uncovered: 1) that the identity of the available catecholamine precursor alters the structure of melanin pigments produced either in Cryptococcus neoformans fungal cells or under cell-free conditions; 2) that the identity of the available precursor alters the scaffold organization and membrane lipid content of melanized fungal cells; 3) that the fungal cells are melanized preferentially by an l-DOPA precursor; and 4) that the macromolecular carbon- and nitrogen-based architecture of cell-free and fungal eumelanins includes indole, pyrrole, indolequinone, and open-chain building blocks that develop depending on reaction time. In conclusion, the availability of catecholamine precursors plays an important role in eumelanin development by affecting the efficacy of pigment formation, the melanin molecular structure, and its underlying scaffold in fungal systems.
Assuntos
Cryptococcus neoformans/metabolismo , Levodopa/metabolismo , Melaninas/biossíntese , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Cryptococcus neoformans/química , Levodopa/química , Melaninas/químicaRESUMO
Density functional theory was employed for a comprehensive study that provided electronic and structural insights into the KatG catalase reaction that involves oxyheme. The catalytic role of a unique amino acid cofactor Met-Tyr-Trp (MYW) in its radical form found in KatG was thereby elucidated. It was established that the MYW-radical is flexible such that a "hinge-like opening" rotation of the Trp-107 ring with respect to the Tyr-229 ring along their covalent C-C bond is an inherent feature of its catalytic properties. Also, an H-bond between the Tyr-229 and the mobile side chain of Arg-418 further enables the catalytic events. The opening process breaks an H-bond between the N-H of Trp-107 and the inner oxygen of the Fe-O2 (oxyheme) complex present in the closed conformation of the MYW-radical. This motion lowers the spin-crossing energy barrier between the ground state and the catalytically active high-spin states and enables electron transfer from the oxyheme group to the MYW-radical. The release of molecular oxygen is thereby catalyzed and leaves ferric-heme poised for another catalytic cycle. The energy barrier for the oxyheme state to complete the catalytic event, when assisted by the radical opening process, is thereby reduced and estimated to be 5.6 kcal/mol.
Assuntos
Proteínas de Bactérias/química , Catalase/química , Heme/química , Metionina/química , Oxigênio/química , Triptofano/química , Tirosina/química , Carbono/química , Catálise , Elétrons , Hidrogênio/química , Ligação de Hidrogênio , Modelos Químicos , Mycobacterium tuberculosis , Nitrogênio/química , Teoria QuânticaRESUMO
We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7, this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities, and oxyferrous state lifetimes, of both heme binding sites in HP7. We find that despite the identical sequence of helices in both binding sites, there are differences in oxygen affinity and oxyferrous state lifetime that may be the result of differences in the freedom of motion imposed by the candelabra fold on the two sites of the protein. We further examine the effect of mutational removal of the buried glutamates on function. Heme iron in the ferrous state of this mutant is rapidly oxidized when exposed to oxygen. Compared to that of HP7, the distal histidine affinity is increased by a 22-fold decrease in the histidine ligand off rate. Electron paramagnetic resonance comparison of these ferric hemoproteins demonstrates that the mutation increases the level of disorder at the heme binding site. Nuclear magnetic resonance-detected deuterium exchange demonstrates that the mutation greatly increases the degree of penetration of water into the protein core. The inability of the mutant protein to bind oxygen may be due to an increased level of water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors. Together, these data underline the importance of the control of protein dynamics in the design of functional artificial proteins.
Assuntos
Globinas/química , Globinas/metabolismo , Oxigênio/metabolismo , Sítios de Ligação , Monóxido de Carbono/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Globinas/genética , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Heme/química , Heme/metabolismo , Histidina/química , Histidina/metabolismo , Cinética , Ligantes , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Água/análiseRESUMO
Catalase activity of the dual-function heme enzyme catalase-peroxidase (KatG) depends on several structural elements, including a unique adduct formed from covalently linked side chains of three conserved amino acids (Met-255, Tyr-229, and Trp-107, Mycobacterium tuberculosis KatG numbering) (MYW). Mutagenesis, electron paramagnetic resonance, and optical stopped-flow experiments, along with calculations using density functional theory (DFT) methods revealed the basis of the requirement for a radical on the MYW-adduct, for oxyferrous heme, and for conserved residues Arg-418 and Asp-137 in the rapid catalase reaction. The participation of an oxyferrous heme intermediate (dioxyheme) throughout the pH range of catalase activity is suggested from our finding that carbon monoxide inhibits the activity at both acidic and alkaline pH. In the presence of H(2)O(2), the MYW-adduct radical is formed normally in KatG[D137S] but this mutant is defective in forming dioxyheme and lacks catalase activity. KatG[R418L] is also catalase deficient but exhibits normal formation of the adduct radical and dioxyheme. Both mutants exhibit a coincidence between MYW-adduct radical persistence and H(2)O(2) consumption as a function of time, and enhanced subunit oligomerization during turnover, suggesting that the two mutations disrupting catalase turnover allow increased migration of the MYW-adduct radical to protein surface residues. DFT calculations showed that an interaction between the side chain of residue Arg-418 and Tyr-229 in the MYW-adduct radical favors reaction of the radical with the adjacent dioxyheme intermediate present throughout turnover in WT KatG. Release of molecular oxygen and regeneration of resting enzyme are thereby catalyzed in the last step of a proposed catalase reaction.
Assuntos
Proteínas de Bactérias/química , Catalase/química , Radicais Livres/química , Mycobacterium tuberculosis/enzimologia , Motivos de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Monóxido de Carbono/química , Catalase/antagonistas & inibidores , Catalase/genética , Domínio Catalítico , Heme/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Químicos , Mutagênese Sítio-Dirigida , Oxigênio/química , Multimerização Proteica , Teoria QuânticaRESUMO
The "acid doping" of a methyl-capped aniline trimer, N-[4-(dimethylamino)phenyl]-N-(4-{[4-(dimethylamino)phenyl]imino}-2,5- cyclohexadien-1-ylidene)-amine (TANI), was performed stoichiometrically to study the nature of charge carriers induced by the acid protonation process. The redox centers in TANI were found to undergo a reversible three-step protonation with 1 equiv, 2 equiv and a large molar excess of dodecylbenzenesulfonic acid (DBSA) in chloroform, as evidenced by three different chromophores (doping levels I, II and III) observed using UV-vis-NIR. Acidity of the dopants and solvent polarity were controlling factors. As revealed by electron paramagnetic resonance spectroscopy (EPR), the doping levels I, II, and III achieved by doping 0.1 mM TANI/chloroform solutions with different amounts of DBSA exhibited relative spin densities of 1:1.2:2.2. Since the expected maximum spin population of TANI through acid doping is two spins per molecule, the reduced paramagnetism given by the doubly protonated TANI (doping level II) indicated partially coupled unpaired spins. The third protonation step (doping level III) produced almost double the unpaired spin concentration compared to the lower doping levels and a multiline EPR spectrum likely comprising two overlapping signals of similar overall line width. The hyperfine couplings contributing to the splittings in this signal were estimated by simulation incorporating 6-H and 1-N nuclei most likely from one highly localized unpaired spin on a terminal dimethylamino group, with an underlying apparent singlet arising from a delocalized unpaired spin; the diradical proposed as the species exhibiting the multiplet EPR signal is isolated by the bridging ammonium cation created by the third doping step. The phenomena suggested the stepwise evolution of partly formed diamagnetic bipolarons from polaron interactions at doping level II and the transformation to the more isolated unsymmetrical system we label "two polarons on a chain" in a triplet state at doping level III. The results provide the characterization of novel doping behaviors for a trimeric aniline molecule in organic solution.
RESUMO
Hemocyanin (Hc) is an oxygen carrier protein in which oxygen binding is regulated by allosteric effectors such as H(+) and L-lactate. Isothermal titration calorimetric measurements showed that L-lactate binds to dodecameric and heterohexameric Hc and to the CaeSS3 homohexamer but not to the CaeSS2 monomer. The binding of lactate caused no change in the optical absorption and x-ray absorption spectra of either oxy- or deoxy-Hc, suggesting that no structural rearrangement of the active site occurred. At pH 6.5, the oxygen binding rate constant k(obs) obtained by flash photolysis showed a significant increase upon addition of L-lactate, whereas L-lactate addition had little effect at pH 8.3. Lactate binding caused a concentration-dependent shift in the interhexameric distances at pH 6.5 based on small angle x-ray scattering measurements. These results show that L-lactate affects oxygen affinity at pH 6.5 by modulating the global structure of Hc without affecting its binuclear copper center (the active site). In contrast to this, the active site structure of deoxy-Hc is affected by changes in pH (Hirota, S., Kawahara, T., Beltramini, M., Di Muro, P., Magliozzo, R. S., Peisach, J., Powers, L. S., Tanaka, N., Nagao, S., and Bubacco, L. (2008) J. Biol. Chem. 283, 31941-31948). Upon addiction of lactate, the kinetic behavior of oxygen rebinding for Hc was heterogeneous under low oxygen concentrations at pH 6.5 due to changes in the T and R state populations, and the equilibrium was found to shift from the T toward the R state with addition of lactate.
Assuntos
Hemocianinas/química , Lactatos/química , Oxigênio/química , Sítio Alostérico , Animais , Artrópodes , Sítios de Ligação , Transporte Biológico , Calorimetria/métodos , Domínio Catalítico , Concentração de Íons de Hidrogênio , Cinética , Modelos Biológicos , Ligação Proteica , Espectroscopia por Absorção de Raios X/métodosRESUMO
A transient tyrosyl-like radical with a narrow doublet X-band EPR signal is present during catalase turnover by Mycobacterium tuberculosis catalase-peroxidase (KatG). Labeling of KatG with beta-methylene-deuterated tyrosine causes a collapse of the doublet to a singlet, while for 3,5-ring-deuterated tyrosine-labeled enzyme, no changes occur in the EPR signal. Except for the replacement Tyr229Phe, all other single-tyrosine mutants of KatG exhibit the same narrow doublet EPR signal and catalase activity similar to that of the wild-type enzyme. These findings confirm that this catalytically competent radical is associated with Tyr229, whose 3' and 5' protons are replaced as a result of cross-links with neighboring Met255 and Trp107 side chains in the post-translationally modified enzyme containing a distal-side Met255-Tyr229-Trp107 adduct.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catalase/metabolismo , Mutagênese Sítio-Dirigida , Peroxidases/química , Peroxidases/metabolismo , Proteínas de Bactérias/genética , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Radicais Livres/metabolismo , Marcação por Isótopo , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Peroxidases/genética , Conformação ProteicaRESUMO
A new approach, the immuno-spin trapping assay, used a novel rabbit polyclonal anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antiserum to detect protein radical-derived DMPO nitrone adducts in the hemoprotein Mycobacterium tuberculosis catalase-peroxidase (KatG). This work demonstrates that the formation of protein nitrone adducts is dependent on the concentrations of tert-BuOOH and DMPO as shown by Western blotting and an enzyme-linked immunosorbent assay (ELISA). We have also detected protein-protein cross-links formed during turnover of Mtb KatG driven by tert-butyl peroxide ( tert-BuOOH) or enzymatic generation of hydrogen peroxide. DMPO inhibits this dimerization due to its ability to trap the amino acid radicals responsible for the cross-linkage. Chemical modifications by tyrosine and tryptophan blockage suggest that tyrosine residues are one site of formation of the dimers. The presence of the tuberculosis drug isoniazid (INH) also prevented cross-linking as a result of its competition for the protein radical. Protein-DMPO nitrone adducts were also generated by a continuous flux of hydrogen peroxide. These findings demonstrated that the protein-based radicals were formed not only during Mtb KatG turnover with alkyl peroxides but also in the presence of hydrogen peroxide. Furthermore, the formation of protein-DMPO nitrone adducts was accelerated in the presence of isoniazid.
Assuntos
Proteínas de Bactérias/química , Catalase/química , Mycobacterium tuberculosis/enzimologia , Detecção de Spin , Animais , Antituberculosos/farmacologia , Óxidos N-Cíclicos/química , Radicais Livres/química , Peróxido de Hidrogênio/farmacologia , Isoniazida/farmacologia , Óxidos de Nitrogênio , Oxidantes/farmacologia , Coelhos , Marcadores de SpinRESUMO
The catalase-peroxidase (KatG) of Mycobacterium tuberculosis (Mtb) is important for the virulence of this pathogen and also is responsible for activation of isoniazid (INH), an antibiotic in use for over 50 years in the first line treatment against tuberculosis infection. Overexpressed Mtb KatG contains a heterogeneous population of heme species that present distinct spectroscopic properties and, as described here, functional properties. A six-coordinate (6-c) heme species that accumulates in the resting enzyme after purification is defined as a unique structure containing weakly associated water on the heme distal side. We present the unexpected finding that this form of the enzyme, generally present as a minority species along with five-coordinate (5-c) enzyme, is the favored reactant for ligand binding. The use of resting enzyme samples with different proportional composition of 5-c and 6-c forms, as well as the use of KatG mutants with replacements at residue 315 that have different tendencies to stabilize the 6-c form, allowed demonstration of more rapid cyanide binding and preferred peroxide binding to enzyme containing 6-c heme. Optical-stopped flow and equilibrium titrations of ferric KatG with potassium cyanide reveal complex behavior that depends in part on the amount of 6-c heme in the resting enzymes. Resonance Raman and low-temperature EPR spectroscopy clearly demonstrate favored ligand (cyanide or peroxide) binding to 6-c heme. The 5-c and 6-c enzyme forms are not in equilibrium on the time scale of the experiments. The results provide evidence for the likely participation of specific water molecule(s) in the first phases of the reaction mechanism of catalase-peroxidase enzymes.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cianetos/metabolismo , Compostos Férricos/metabolismo , Mycobacterium tuberculosis/enzimologia , Peroxidases/química , Peroxidases/metabolismo , Água/metabolismo , Proteínas de Bactérias/genética , Espectroscopia de Ressonância de Spin Eletrônica , Isomerismo , Ligantes , Mutação , Peroxidases/genética , Prótons , Análise Espectral Raman , TitulometriaRESUMO
Mycobacterium tuberculosis catalase-peroxidase (Mtb KatG) is a bifunctional enzyme that possesses both catalase and peroxidase activities and is responsible for the activation of the antituberculosis drug isoniazid. Mtb KatG contains an unusual adduct in its distal heme-pocket that consists of the covalently linked Trp107, Tyr229, and Met255. The KatG(Y229F) mutant lacks this adduct and has decreased steady-state catalase activity and enhanced peroxidase activity. In order to test a potential structural role of the adduct that supports catalase activity, we have used resonance Raman spectroscopy to probe the local heme environment of KatG(Y229F). In comparison to wild-type KatG, resting KatG(Y229F) contains a significant amount of 6-coordinate, low-spin heme and a more planar heme. Resonance Raman spectroscopy of the ferrous-CO complex of KatG(Y229F) suggest a non-linear Fe-CO binding geometry that is less tilted than in wild-type KatG. These data provide evidence that the Met-Tyr-Trp adduct imparts structural stability to the active site of KatG that seems to be important for sustaining catalase activity.
Assuntos
Proteínas de Bactérias/química , Catalase/química , Tirosina/química , Substituição de Aminoácidos , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catalase/metabolismo , Reagentes de Ligações Cruzadas/química , Espectroscopia de Ressonância de Spin Eletrônica , Isoniazida/metabolismo , Isoniazida/farmacologia , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/enzimologia , Peroxidases/química , Fenilalanina/química , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise Espectral RamanRESUMO
The reaction of Mycobacterium tuberculosis KatG and the mutant KatG(S315T) with two different organic peroxides is studied using resonance Raman spectroscopy. For the first time, an intermediate is observed in a catalase-peroxidase with vibrations that are characteristic of Compound II. The observation of this intermediate is consistent with photoreduction of Compound I and is in agreement with the formation of Compound I during the catalytic cycle of KatG. The same intermediate is detected in KatG(S315T), a mutant associated with resistance to isoniazid (INH), but with a lower yield, indicating that the organic peroxides cannot react with the heme iron in KatG(S315T) as efficiently as in wild-type KatG. Our results are consistent with catalytic competence of the S315T mutant and support the model that the S315T mutation confers antibiotic resistance by modifying the interaction between the enzyme and the drug.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catalase/genética , Catalase/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Peróxidos/química , Peróxidos/metabolismo , Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Genes Bacterianos , Ferro/química , Ferro/metabolismo , Isoniazida/farmacologia , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Oxirredução , Fotoquímica , Análise Espectral RamanRESUMO
A single amino acid mutation (W321F) in Mycobacterium tuberculosis catalase-peroxidase (KatG) was constructed by site-directed mutagenesis. The purified mutant enzyme was characterized using optical and electron paramagnetic resonance spectroscopy, and optical stopped-flow spectrophotometry. Reaction of KatG(W321F) with 3-chloroperoxybenzoic acid, peroxyacetic acid, or t-butylhydroperoxide showed formation of an unstable intermediate assigned as Compound I (oxyferryl iron:porphyrin pi-cation radical) by similarity to wild-type KatG, although second-order rate constants were significantly lower in the mutant for each peroxide tested. No evidence for Compound II was detected during the spontaneous or substrate-accelerated decay of Compound I. The binding of isoniazid, a first-line anti-tuberculosis pro-drug activated by catalase-peroxidase, was noncooperative and threefold weaker in KatG(W321F) compared with wild-type enzyme. An EPR signal assigned to a protein-based radical tentatively assigned as tyrosyl radical in wild-type KatG, was also observed in the mutant upon reaction of the resting enzyme with alkyl peroxide. These results show that mutation of residue W321 in KatG does not lead to a major alteration in the identity of intermediates formed in the catalytic cycle of the enzyme in the time regimes examined here, and show that this residue is not the site of stabilization of a radical as might be expected based on homology to yeast cytochrome c peroxidase. Furthermore, W321 is indicated to be important in KatG for substrate binding and subunit interactions within the dimer, providing insights into the origin of isoniazid resistance in clinically isolated KatG mutants.
Assuntos
Proteínas de Bactérias , Mutação , Mycobacterium tuberculosis/enzimologia , Peroxidases/química , Sequência de Aminoácidos , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/metabolismo , Cinética , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos/metabolismo , Transdução de Sinais , Espectrofotometria , Fatores de TempoRESUMO
Peroxidatic activation of the anti-tuberculosis pro-drug isoniazid by Mycobacterium tuberculosis catalase-peroxidase (KatG) is regulated by gating residues of a heme access channel. The steric restriction at the bottleneck of this channel is alleviated by replacement of residue Asp137 with Ser, according to crystallographic and kinetic studies.
Assuntos
Antituberculosos/metabolismo , Proteínas de Bactérias/metabolismo , Catalase/metabolismo , Isoniazida/metabolismo , Mycobacterium tuberculosis/enzimologia , Pró-Fármacos/metabolismo , Proteínas de Bactérias/química , Catalase/química , Humanos , Modelos Moleculares , Tuberculose/microbiologiaRESUMO
The synthesis and characterization of a new water-soluble N,N-chelating iminophosphorane ligand TPAN-C(O)-2-NC(5)H(4) (N,N-IM) (1) and its d(8) (Au(III), Pd(II) and Pt(II)) coordination complexes are reported. The structures of cationic [AuCl(2)(N,N-IM)]ClO(4) (2) and neutral [MCl(2)(N,N-IM)] M=Pd (3), Pt(4) complexes were determined by X-ray diffraction studies or by means of density-functional calculations. While the Pd and Pt compounds are stable in mixtures of DMSO/H(2)O over 4 days, the gold derivative (2) decomposes quickly to TPAO and previously reported neutral gold(III) compound [AuCl(2)(N,N-H)] 5 (containing the chelating N,N-fragment HN-C(O)-2-NC(5)H(4)). The cytotoxicities of complexes 2-5 were evaluated in vitro against human Jurkat-T acute lymphoblastic leukemia cells and DU-145 human prostate cancer cells. Pt (4) and Au compounds (2 and 5) are more cytotoxic than cisplatin to these cell lines and to cisplatin-resistant Jurkat sh-Bak cell lines and their cell death mechanism is different from that of cisplatin. All the compounds show higher toxicity against leukemia cells when compared to normal human T-lymphocytes (PBMC). The interaction of the Pd and Pt compounds with calf thymus and plasmid (pBR322) DNA is different from that of cisplatin. All compounds bind to human serum albumin (HSA) faster than cisplatin (measured by fluorescence spectroscopy). Weak and stronger binding interactions were found for the Pd (3) and Pt (4) derivatives by isothermal titration calorimetry. Importantly, for the Pt (4) compounds the binding to HSA was reversed by addition of a chelating agent (citric acid) and by a decrease in pH.
Assuntos
Antineoplásicos/química , DNA/química , Metais/química , Fosforanos/química , Albumina Sérica/química , Linhagem Celular Tumoral , Dicroísmo Circular , Cisplatino/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
The objective of this study was to determine the effect of (bi)sulfite (hydrated sulfur dioxide) on human neutrophils and the ability of these immune cells to produce reactive free radicals due to (bi)sulfite oxidation. Myeloperoxidase (MPO) is an abundant heme protein in neutrophils that catalyzes the formation of cytotoxic oxidants implicated in asthma and inflammatory disorders. In this study sulfite ((â¢)SO(3)(-)) and sulfate (SO(4)(â¢-)) anion radicals are characterized with the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the reaction of (bi)sulfite oxidation by human MPO and human neutrophils via sulfite radical chain reaction chemistry. After treatment with (bi)sulfite, phorbol 12-myristate 13-acetate-stimulated neutrophils produced DMPO-sulfite anion radical, -superoxide, and -hydroxyl radical adducts. The last adduct probably resulted, in part, from the conversion of DMPO-sulfate to DMPO-hydroxyl radical adduct via a nucleophilic substitution reaction of the radical adduct. This anion radical (SO(4)(â¢-)) is highly reactive and, presumably, can oxidize target proteins to protein radicals, thereby initiating protein oxidation. Therefore, we propose that the potential toxicity of (bi)sulfite during pulmonary inflammation or lung-associated diseases such as asthma may be related to free radical formation.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Radicais Livres/metabolismo , Ativação de Neutrófilo , Neutrófilos/metabolismo , Sulfitos/metabolismo , Células Cultivadas , Humanos , Neutrófilos/enzimologia , Oxirredução , Peroxidase/antagonistas & inibidores , Peroxidase/metabolismo , Marcadores de SpinRESUMO
The interactions of π-arene-Ru(II)-chloroquine complexes with human serum albumin (HSA), apotransferrin and holotransferrin have been studied by circular dichroism (CD) and UV-Visible spectroscopies, together with isothermal titration calorimetry (ITC). The data for [Ru(η(6)-p-cymene)(CQ)(H(2)O)Cl]PF(6) (1), [Ru(η(6)-benzene)(CQ)(H(2)O)Cl]PF(6) (2), [Ru(η(6)-p-cymene)(CQ)(H(2)O)(2)][PF(6)](2) (3), [Ru(η(6)-p-cymene)(CQ)(en)][PF(6)](2) (4), [Ru(η(6)-p-cymene)(η(6)-CQDP)][BF(4)](2) (5) (CQ: chloroquine; DP: diphosphate; en: ethylenediamine), in comparison with CQDP and [Ru(η(6)-p-cymene)(en)Cl][PF(6)] (6) as controls demonstrate that 1, 2, 3, and 5, which contain exchangeable ligands, bind to HSA and to apotransferrin in a covalent manner. The interaction did not affect the α-helical content in apotransferrin but resulted in a loss of this type of structure in HSA. The binding was reversed in both cases by a decrease in pH and in the case of the Ru-HSA adducts, also by addition of chelating agents. A weaker interaction between complexes 4 and 6 and HSA was measured by ITC but was not detectable spectroscopically. No interactions were observed for complexes 4 and 6 with apotransferrin or for CQDP with either protein. The combined results suggest that the arene-Ru(II)-chloroquine complexes, known to be active against resistant malaria and several lines of cancer cells, also display a good transport behavior that makes them good candidates for drug development.
Assuntos
Antimaláricos/química , Antineoplásicos/química , Cloroquina/química , Compostos Organometálicos/química , Rutênio/química , Albumina Sérica/química , Transferrina/química , Apoproteínas/química , Calorimetria , Dicroísmo Circular , Interações Medicamentosas , Humanos , Estrutura MolecularRESUMO
Certain fungi thrive in highly radioactive environments including the defunct Chernobyl nuclear reactor. Cryptococcus neoformans (C. neoformans), which uses L-3,4-dihydroxyphenylalanine (L-DOPA) to produce melanin, was used here to investigate how gamma radiation under aqueous aerobic conditions affects the properties of melanin, with the aim of gaining insight into its radioprotective role. Exposure of melanized fungal cell in aqueous suspensions to doses of γ-radiation capable of killing 50 to 80% of the cells did not lead to a detectable loss of melanin integrity according to EPR spectra of melanin radicals. Moreover, upon UV-visible (Xe-lamp) illumination of melanized cells, the increase in radical population was unchanged after γ-irradiation. Gamma-irradiation of frozen cell suspensions and storage of samples for several days at 77 K however, produced melanin modification noted by a reduced radical population and reduced photoresponse. More direct evidence for structural modification of melanin came from the detection of soluble products with absorbance maxima near 260 nm in supernatants collected after γ-irradiation of cells and cell-free melanin. These products, which include thiobarbituric acid (TBA)-reactive aldehydes, were also generated by Fenton reagent treatment of cells and cell-free melanin. In an assay of melanin integrity based on the metal (Bi(+3)) binding capacity of cells, no detectable loss in binding was detected after γ-irradiation. Our results show that melanin in C. neoformans cells is susceptible to some damage by hydroxyl radical formed in lethal radioactive aqueous environments and serves a protective role in melanized fungi that involves sacrificial breakdown.
Assuntos
Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/efeitos da radiação , Raios gama/efeitos adversos , Melaninas/química , Melaninas/metabolismo , Radical Hidroxila/metabolismoRESUMO
KatG (catalase-peroxidase) in Mycobacterium tuberculosis is responsible for activation of isoniazid (INH), a pro-drug used to treat tuberculosis infections. Resistance to INH is a global health problem most often associated with mutations in the katG gene. The origin of INH resistance caused by the KatG[S315G] mutant enzyme is examined here. Overexpressed KatG[S315G] was characterized by optical, EPR, and resonance Raman spectroscopy and by studies of the INH activation mechanism in vitro. Catalase activity and peroxidase activity with artificial substrates were moderately reduced (50 and 35%, respectively), whereas the rates of formation of oxyferryl heme:porphyrin pi-cation radical and the decay of heme intermediates were approximately 2-fold faster in KatG[S315G] compared with WT enzyme. The INH binding affinity for the resting enzyme was unchanged, whereas INH activation, measured by the rate of formation of an acyl-nicotinamide adenine dinucleotide adduct considered to be a bactericidal molecule, was reduced by 30% compared with WT KatG. INH resistance is suggested to arise from a redirection of catalytic intermediates into nonproductive reactions that interfere with oxidation of INH. In the resting mutant enzyme, a rapid evolution of 5-c heme to 6-c species occurred in contrast with the behavior of WT KatG and KatG[S315T] and consistent with greater flexibility at the heme edge in the absence of the hydroxyl of residue 315. Insights into the effects of mutations at residue 315 on enzyme structure, peroxidation kinetics, and specific interactions with INH are presented.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Catalase/genética , Farmacorresistência Bacteriana/fisiologia , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/metabolismo , Calorimetria , Catalase/metabolismo , Ativação Enzimática/genética , Escherichia coli , Técnicas In Vitro , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/genética , Peroxidases/metabolismo , Análise Espectral Raman , Especificidade por Substrato , Temperatura , TitulometriaRESUMO
Catalase-peroxidase (KatG) is essential in Mycobacterium tuberculosis for oxidative stress management and activation of the antitubercular pro-drug isoniazid. The role of a unique distal side adduct found in KatG enzymes, involving linked side chains of residues Met255, Tyr229, and Trp107 (MYW), in the unusual catalase activity of KatG is addressed here and in our companion paper (Suarez, J., Ranguelova, K., Jarzecki, A. A., Manzerova, J., Krymov, V., Zhao, X., Yu, S., Metlitsky, L., Gerfen, G. J., and Magliozzo, R. S. (2009) J. Biol. Chem. 284, in press). The KatG[W107F] mutant exhibited severely reduced catalase activity yet normal peroxidase activity, and as isolated contains more abundant 6-coordinate heme in high spin and low spin forms compared with the wild-type enzyme. Most interestingly, oxyferrous heme is also found in the purified enzyme. Oxyferrous KatG[W107F] was prepared by photolysis in air of the carbonyl enzyme or was generated using hydrogen peroxide decayed with a t1/2 of 2 days compared with 6 min for wild-type protein. The stability of oxyenyzme was modestly enhanced in KatG[Y229F] but was not affected in KatG[M255A]. Optical stopped-flow experiments showed rapid formation of Compound I in KatG[W107F] and facile formation of oxyferrous heme in the presence of micromolar hydrogen peroxide. An analysis of the relationships between catalase activity, stability of oxyferrous enzyme, and a proposed MYW adduct radical is presented. The loss of catalase function is assigned to the loss of the MYW adduct radical and structural changes that lead to greatly enhanced stability of oxyenzyme, an intermediate of the catalase cycle of native enzyme.