RESUMO
A subfamily of rhodopsin pigments was recently discovered in bacteria and proposed to function as dual-function light-driven H(+)/Na(+) pumps, ejecting sodium ions from cells in the presence of sodium and protons in its absence. This proposal was based primarily on light-induced proton flux measurements in suspensions of Escherichia coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the putative Na(+) and H(+) transport definitively. We expressed IAR, an uncharacterized member from Indibacter alkaliphilus in E. coli cell suspensions, and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na(+) pumping activity in vitro, namely, light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na(+)-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore. These results meet the criteria for electrogenic Na(+) transport and electrogenic H(+) transport, respectively, in the presence and absence of Na(+). The direction of proton fluxes indicated that IAR was inserted inside-out into our sealed LUV system, which we confirmed by site-directed spin-label electron paramagnetic resonance spectroscopy. We further demonstrate that Na(+) transport by IAR requires Na(+) only on the cytoplasmic side of the protein. The in vitro LUV system proves that the dual light-driven H(+)/Na(+) pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo.
Assuntos
Luz , Prótons , Rodopsinas Microbianas/metabolismo , Rodopsinas Microbianas/efeitos da radiação , Sódio/metabolismo , Escherichia coli , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Lasers , Fotólise , Análise Espectral , Lipossomas UnilamelaresRESUMO
A recently discovered rhodopsin ion pump (DeNaR, also known as KR2) in the marine bacterium Dokdonia eikasta uses light to pump protons or sodium ions from the cell depending on the ionic composition of the medium. In cells suspended in a KCl solution, DeNaR functions as a light-driven proton pump, whereas in a NaCl solution, DeNaR conducts light-driven sodium ion pumping, a novel activity within the rhodopsin family. These two distinct functions raise the questions of whether the conformations of the protein differ in the presence of K(+) or Na(+) and whether the helical movements that result in the canonical E â C conformational change in other microbial rhodopsins are conserved in DeNaR. Visible absorption maxima of DeNaR in its unphotolyzed (dark) state show an 8 nm difference between Na(+) and K(+) in decyl maltopyranoside micelles, indicating an influence of the cations on the retinylidene photoactive site. In addition, electronic paramagnetic resonance (EPR) spectra of the dark states reveal repositioning of helices F and G when K(+) is replaced with Na(+). Furthermore, the conformational changes assessed by EPR spin-spin dipolar coupling show that the light-induced transmembrane helix movements are very similar to those found in bacteriorhodopsin but are altered by the presence of Na(+), resulting in a new feature, the clockwise rotation of helix F. The results establish the first observation of a cation switch controlling the conformations of a microbial rhodopsin and indicate specific interactions of Na(+) with the half-channels of DeNaR to open an appropriate path for ion translocation.
Assuntos
Proteínas de Bactérias/metabolismo , Cátions/metabolismo , Flavobacteriaceae/metabolismo , Potássio/metabolismo , Bombas de Próton/metabolismo , Rodopsina/metabolismo , Sódio/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cátions/química , Flavobacteriaceae/química , Flavobacteriaceae/genética , Modelos Moleculares , Potássio/química , Conformação Proteica , Bombas de Próton/química , Bombas de Próton/genética , Rodopsina/química , Rodopsina/genética , Sódio/químicaRESUMO
The accumulation of plagues of ß-amyloid (Aß) peptides in the brain is a hallmark of Alzheimer's disease (AD). The redox-active Cu and Fe complexes of Aß can cause damage to the neurons potentially via reactive oxygen species (ROS). The significant metal-mediated oxidative activity of CuAß suggests that its presence can be chemically devastating regardless whether it is a cause or a result of AD. Flavonoids exhibit various benefits to human health, attributable to their metal-binding and antioxidation activities to certain extents. Despite broad interests and extensive studies of their metal-binding properties and anti/pro-oxidation activities, these properties and the mechanisms of the activities toward metal-centered oxidation reactions have not been fully revealed and concluded. We report herein distinctive antioxidation mechanisms between two flavonoid families toward the oxidation reactions by CuAß(1-20), wherein the flavonols quercetin (Qr) and myricetin (Mr) competitively inhibit the oxidation of catechol by CuAß(1-20) with K(i) of 11.2 and 32.6 µM, respectively, whereas the flavanols catechin (Ct) and epicatechin (Et) are substrates with k(cat) = 1.01 × 10(-2) and 1.55 × 10(-3) s(-1) and K(m) = 0.94 and 0.55 mM, respectively. Qr has a nearly 10-fold higher antioxidative efficacy than Ct against the oxidation activity of CuAß, while Ct is effectively oxidized, which further decreases its antioxidant capacity. Similar inhibition patterns are observed toward oxidation of the catecholamine neurotransmitter dopamine by CuAß(1-20). Metal ions and CuAß bind Qr with a 1:1 ratio under our experimental conditions through the α-ketoenolate moiety as determined by the use of Co(2+) and Yb(3+) as paramagnetic NMR probes. Unlike flavanols, which are merely suicide antioxidative substrates, flavonols bind to the metal center and prevent metal-mediated redox reactions. We suggest flavonols may serve as leads for drug discovery and/or as agents toward preventing metal-mediated oxidative stress due to AD and other disorders. Moreover, CuAß shows 8.6- and 4.2-fold higher kinetic regioselectivity in terms of k(cat) and k(cat)/K(m), respectively, toward the peroxidation of Ct than that of the enantiomer Et, suggesting potential development of metallo-catalysts in regioselective catalysis by the use of metallopeptides as templates.
Assuntos
Peptídeos beta-Amiloides/química , Antioxidantes/química , Complexos de Coordenação/química , Cobre/química , Flavonoides/química , Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Antioxidantes/metabolismo , Complexos de Coordenação/metabolismo , Cobre/metabolismo , Flavonoides/metabolismo , Flavonoides/farmacologia , Cinética , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Oxirredução , Quercetina/química , Quercetina/metabolismoRESUMO
Adrenal cytochrome b(561) (AdCytb) is the prototype of a widespread protein family that specializes in delivering electrons donated by ascorbic acid for different processes in eukaryotic cells. AdCytb transports redox equivalents from cytoplasmic ascorbate across the membranes of chromaffin granules to support norepinephrine synthesis within their matrix. The interaction of AdCytb with ascorbate is central to a proposed mechanism of AdCytb's function, and a histidine in the active site of AdCytb was suggested to bind cytoplasmic ascorbate and serve as the acceptor of the proton released during ascorbate oxidation. AdCytb contains high- and low-potential hemes but their orientation relative to the matrix and cytoplasmic interfaces of chromaffin granule membrane is disputed. Using a combination of three spectroscopic methods (UV-vis absorption, near-infrared magnetic circular dichroism, and electron paramagnetic resonance), we find that a histidine residue that serves as an axial ligand to the high-potential heme undergoes deprotonation with a pK of ~8.0 and is thus a good candidate for interaction with cytoplasmic ascorbate. The low-potential heme of AdCytb is found to have a pK of ~10.5, making it an unlikely candidate for accepting a proton at physiological pH. UV-vis spectroscopy reveals an additional proton acceptor group in AdCytb with a pK of ~6.5 that is not observed by the other two techniques; whether it plays a role in the mechanism of AdCytb is unknown. We incorporate these results into an updated mechanism of AdCytb reduction predicated on the high-potential heme's localization on the cytoplasmic interface of the chromaffin granule membrane.
Assuntos
Citocromos b/metabolismo , Glândulas Suprarrenais/metabolismo , Ácido Ascórbico/metabolismo , Grânulos Cromafim/metabolismo , Citocromos b/química , Espectroscopia de Ressonância de Spin Eletrônica , Heme/metabolismo , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Oxirredução , Análise EspectralRESUMO
Several residues in the third extramembrane segment (EM3) of adrenal cytochrome b(561) have been proposed to be involved in this cytochrome's interaction with ascorbate, but there has been no systematic evaluation of residues in the segment. We used alanine scanning mutagenesis to assess the functional and structural roles of the EM3 residues and several adjacent residues (residues 70-85) in the bovine cytochrome. Each alanine mutant was expressed in a bacterial system, solubilized with detergent, and affinity-purified. The recombinant proteins contained approximately two hemes per monomer and, except for R74A, retained basic functionality (≥ 94% reduced by 20 mM ascorbate). Equilibrium spectrophotometric titrations with ascorbate were used to analyze the α-band line shape and amplitude during reduction of the high- and low-potential heme centers (b(H) and b(L), respectively) and the midpoint ascorbate concentrations for the b(H) and b(L) transitions (C(H) and C(L), respectively). Y73A and K85A markedly narrowed the b(H) α-band peak; other mutants had weaker effects or no effect on b(H) or b(L) spectra. Relative changes in C(H) for the mutants were larger than changes in C(L), with 1.5-2.9-fold increases in C(H) for L70A, L71A, Y73A, R74A, N78A, and K85A. The amounts of functional b(H) and b(L) centers in additional Arg74 mutants, assessed by ascorbate titration and EPR spectroscopy, declined in concert in the following order: wild type > R74K > R74Q > R74T and R74Y > R74E. The results of this first comprehensive experimental test of the proposed roles of EM3 residues have identified residues with a direct or indirect impact on ascorbate interactions, on the environment of the b(H) heme center, and on formation of the native b(H)-b(L) unit. Surprisingly, no individual EM3 residue was by itself indispensable for the interaction with ascorbate, and the role of the segment appears to be more subtle than previously thought. These results also support our topological model of the adrenal cytochrome, which positions b(H) near the cytoplasmic side of the membrane.
Assuntos
Glândulas Suprarrenais/enzimologia , Membrana Celular/metabolismo , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Alanina , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Ácido Ascórbico/metabolismo , Bovinos , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/isolamento & purificação , Análise Mutacional de DNA , Heme/metabolismo , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The potential risk of metal-centered oxidative catalysis has been overlooked in the research of the copper complexes of the Alzheimer's disease-related beta-amyloid (Abeta) peptides. Cu(2+) complexes of Abeta(1-40) and its 1-16 and 1-20 fragments have recently been shown to exhibit significant metal-centered oxidative activities toward several catecholamine neurotransmitters with and without H(2)O(2) around neutral pH [G.F.Z. da Silva, L.-J. Ming, "Metallo-ROS" in Alzheimer's disease: metal-centered oxidation of neurotransmitters by Cu(II)-beta-amyloid and neuropathology of Alzheimer's disease, Angew. Chem. Int. Ed. 46 (2007) 3337-3341]. The results further support the metallo-Abeta-associated oxidative stress theory often considered to be connected to the neuropathology of the disease. The metal-centered oxidative catalysis of CuAbeta(1-16/20) challenges the long-standing proposed redox role of Met35 in Abeta because Abeta(1-16/20) do not contain a Met. External Met has been determined by kinetic, optical, and electron paramagnetic resonance methods to bind directly to the Cu(2+) center of CuAbeta(1-40) and CuAbeta(1-20) with K(d)=2.8 mM and 11.3 microM, respectively, which reflects less accessibility of the metal center in the full-length CuAbeta(1-40). However, Met does not serve as a reducing agent for the Cu(II) which thus must amplify the observed oxidative catalysis of CuAbeta(1-20)through a non-redox mechanism. Conversely, the CuAbeta-catalyzed oxidation reaction of dopamine is inhibited by bio-available reducing agents such as ascorbate (competitive K(ic)=66 microM) and glutathione (non-competitive, K(inc)=53 microM). These data indicate that the oxidation chemistry of metallo-Abeta is not initiated by Met35. The results yield further molecular and mechanistic insights into the roles of metallo-Abeta in this disease.
Assuntos
Peptídeos beta-Amiloides/química , Cobre/química , Metionina/química , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Sítios de Ligação , Catecol Oxidase/química , Catecol Oxidase/metabolismo , Dopamina/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Técnicas In Vitro , Cinética , Modelos Químicos , Oxirredução , Estresse Oxidativo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Substâncias RedutorasRESUMO
The peptidyl antibiotic bacitracin (Bc) is one of the most widely used antibiotics which can bind divalent transition metal ions, including Mn(II), Co(II), Ni(II), Cu(II), and Zn(II). The metal binding is essential for its antimicrobial activity. Previous analysis of the hyperfine-shifted (1)H NMR signals of Co(II)-Bc A(1) revealed the structure of the metal binding environment and a potential hydrophobic site important for the bioactivity of this antibiotic. Co(II)-Bc in DMSO shows relatively sharper hyperfine-shifted (1)H NMR signals compared with the spectrum acquired in an aqueous solution, allowing more thorough analysis of the signals with 1D and 2D NMR methods. Pyrophosphate and derivatives bind to Co(II)-Bc to form kinetically inert ternary complexes. The coordinated D-Glu-4 is found detached from the metal center of metallobacitracin upon trimetaphosphate binding, implying its role in the antibiotic activity of Bc. We further demonstrate in this report the structure-function relationship on desamido-Bc of low antibiotic activity by the use of NMR, wherein D-Glu-4 is suggested to be important for the bioactivity of Bc. The interaction of the phospho-moiety with Bc is also reflected by DNA binding, wherein metal-free Bc does not bind DNA, whereas various metal complexes of Bc do. Cu(II)-Bc was further demonstrated to bind and oxidatively cleave DNA under reduction conditions in the air. It also exhibited a significant oxidative activity toward catechol oxidation, showing enzyme-like saturation kinetics with k(cat) = 7.0 x 10(-3) s(-1) and k(cat)/K(m) = 2.1 M(-1) s(-1) aerobically and k(cat) = 0.38 s(-1) and k(cat)/K(m) = 14.7 M(-1) s(-1) in the presence of 32 mM of H(2)O(2). The binding of pyrophosphate moiety to metallobacitracin, the detachment of d-Glu-4, and the significant oxidative activity of Cu(II)-Bc provide further insights into the bioactivity of this metallopeptide and Cu-oxygen chemistry.
Assuntos
Antibacterianos/química , Antibacterianos/metabolismo , Bacitracina/química , Bacitracina/metabolismo , DNA/metabolismo , Difosfatos/metabolismo , Ácido Glutâmico/metabolismo , Cobalto/metabolismo , Cobre/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Oxirredução , Ligação Proteica , Conformação ProteicaRESUMO
Adrenal cytochrome b(561) (cyt b(561)), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b(561) (Bashtovyy, D., Berczi, A., Asard, H., and Pali, T. (2003) Protoplasma 221, 31-40) predicts that His92 is near the His88/His161 heme and that His110 abuts the His54/His122 heme. We tested these predictions by analyzing the effects of mutations at His92 or His110 on the spectroscopic and functional properties. Wild type cytochrome and mutants with substitutions in other histidine residues or in Asn78 were used for comparison. The largest lineshape changes in the optical absorbance spectrum of the high-potential (b(H)) peak were seen with mutation of His92; the largest changes in the low-potential (b(L)) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g=3.1 signal (b(H)) but not the g=3.7 signal (b(L)). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the b(H) transition; mutations in His110 produced the largest decreases in DeltaA(561) for the b(L) transition. These results indicate that His92 can be considered part of the b(H) heme center, and His110 part of the b(L) heme center, in adrenal cyt b(561).
Assuntos
Glândulas Suprarrenais/metabolismo , Grupo dos Citocromos b/metabolismo , Heme/metabolismo , Histidina/metabolismo , Sequência de Aminoácidos , Animais , Ácido Ascórbico/metabolismo , Bovinos , Grupo dos Citocromos b/química , Espectroscopia de Ressonância de Spin Eletrônica , Heme/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Oxirredução , Estrutura Secundária de Proteína , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise Espectral , Relação Estrutura-Atividade , TitulometriaAssuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Cobre/química , Transtornos Neuróticos/patologia , Neurotransmissores/química , Compostos Organometálicos/química , Espécies Reativas de Oxigênio/metabolismo , Peptídeos beta-Amiloides/metabolismo , Catecolaminas/química , Catecóis/química , Cátions Bivalentes , Dopamina/química , Peróxido de Hidrogênio/química , Modelos Químicos , Transtornos Neuróticos/metabolismo , Neurotransmissores/metabolismo , Oxirredução , Oxirredutases/química , Dodecilsulfato de Sódio/químicaAssuntos
Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/química , Catecol Oxidase/química , Cobre/química , Monofenol Mono-Oxigenase/química , Peptídeos beta-Amiloides/metabolismo , Catecol Oxidase/metabolismo , Modelos Biológicos , Estrutura Molecular , Monofenol Mono-Oxigenase/metabolismo , Oxirredução , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismoRESUMO
Blastula protease 10 (BP10) is a metalloenzyme involved in sea urchin embryogenesis, which has been assigned to the astacin family of zinc-dependent endopeptidases. It shows greatest homology with the mammalian tolloid-like genes and contains conserved structural motifs consistent with astacin, tolloid, and bone morphogenetic protein 1. Astacin, a crustacean digestive enzyme, has been proposed to carry out hydrolysis via a metal-centered mechanism that involves a metal-coordinated "tyrosine switch." It has not been determined if the more structurally complex members of this family involved in eukaryotic development share this mechanism. The recombinant BP10 has been overexpressed in Escherichia coli, its metalloenzyme nature has been confirmed, and its catalytic properties have been characterized through kinetic studies. BP10 shows significant hydrolysis toward gelatin both in its native zinc-containing form and copper derivative. The copper derivative of BP10 shows a remarkable 960% rate acceleration toward the hydrolysis of the synthetic substrate N-benzoyl-arginine-p-nitroanilide when compared with the zinc form. The enzyme also shows calcium-dependent activation. These are the first thorough mechanistic studies reported on BP10 as a representative of the more structurally complex members of astacin-type enzymes in deuterostomes, which can add supporting data to corroborate the metal-centered mechanism proposed for astacin and the role of the coordinated Tyr. We have demonstrated the first mechanistic study of a tolloid-related metalloenzyme involved in sea urchin embryogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Metaloendopeptidases/biossíntese , Metaloendopeptidases/química , Metaloproteases/biossíntese , Animais , Benzoilarginina Nitroanilida/química , Cálcio/química , Catálise , Dicroísmo Circular , Clonagem Molecular , Cobre/química , Relação Dose-Resposta a Droga , Elétrons , Eletroforese em Gel de Poliacrilamida , Embrião não Mamífero/metabolismo , Escherichia coli/metabolismo , Gelatinases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Metaloproteases/química , Modelos Químicos , Proteínas Recombinantes/química , Ouriços-do-Mar , Espectrofotometria , Especificidade por Substrato , Fatores de Tempo , Tirosina/química , Zinco/químicaRESUMO
Streptomyces griseus aminopeptidase exhibits activities toward the hydrolyses of peptides and bis(p-nitrophenyl)phosphate (40 billion fold) and catechol oxidation reported herein with catalytic efficiency (kcat/Km) only about 10 times smaller than that of gypsywort catechol oxidase. The multifunctionality of this enzyme suggests that it is a unique system for further exploration of protein structure and function and a template for design of enzymes of diverse activities.
Assuntos
Aminopeptidases/química , Catecol Oxidase/metabolismo , Cobre/química , Streptomyces griseus/enzimologia , Aminopeptidases/metabolismo , Catecol Oxidase/química , Cobre/metabolismo , Modelos MolecularesRESUMO
The Cu2+ complexes of the 1-16 and the 1-20 fragments of the Alzheimer's disease-related beta-amyloid peptide (CuAbeta) show significant oxidative activities toward a catechol-like substrate trihydroxylbenzene and plasmid DNA cleavage. The latter reflects possible oxidative stress to biological macromolecules, yielding supporting data to the pathological role of these soluble Abeta fragments. The former exhibits enzyme-like kinetics and is dependent on [H2O2], exhibiting k(cat) of 0.066 s-1 (6000-fold higher than the reaction without CuAbeta) and k(cat)/Km of 37.2 m-1s-1 under saturating [H2O2] of approximately 0.24%. This kinetic profile is consistent with metal-centered redox chemistry for the action of CuAbeta. A mechanism is proposed by the use of the catalytic cycle of dinuclear catechol oxidase as a working model. Trihydroxylbenzene is also oxidized by CuAbeta aerobically without H2O2, affording rate constants of 6.50x10(-3) s-1 and 3.25 m-1s-1. This activity is also consistent with catechol oxidase action in the absence of H2O2, wherein the substrate binds and reduces the Cu2+ center first, followed by O2 binding to afford the mu-eta2:eta2-peroxo intermediate, which oxidizes a second substrate to complete the catalytic cycle. A tetragonally distorted octahedral metal coordination sphere with three coordinated His side chains and some specific H-bonding interactions is concluded from the electronic spectrum of CuAbeta, hyperfine-shifted 1H NMR spectrum of CoAbeta, and molecular mechanics calculations. The results presented here are expected to add further insight into the chemistry of metallo-Abeta, which may assist better understanding of the neuropathology of Alzheimer's disease.