RESUMO
A growing number of bacteria are recognized to conduct electrons across their cell envelope, and yet molecular details of the mechanisms supporting this process remain unknown. Here, we report the atomic structure of an outer membrane spanning protein complex, MtrAB, that is representative of a protein family known to transport electrons between the interior and exterior environments of phylogenetically and metabolically diverse microorganisms. The structure is revealed as a naturally insulated biomolecular wire possessing a 10-heme cytochrome, MtrA, insulated from the membrane lipidic environment by embedding within a 26 strand ß-barrel formed by MtrB. MtrAB forms an intimate connection with an extracellular 10-heme cytochrome, MtrC, which presents its hemes across a large surface area for electrical contact with extracellular redox partners, including transition metals and electrodes.
Assuntos
Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Proteínas de Ligação a RNA/ultraestrutura , Fatores de Transcrição/ultraestrutura , Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Citocromos/metabolismo , Transporte de Elétrons/fisiologia , Elétrons , Heme/metabolismo , Complexos Multiproteicos/ultraestrutura , Oxirredução , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Inositol polyphosphates are ubiquitous molecular signals in metazoans, as are their pyrophosphorylated derivatives that bear a so-called 'high-energy' phosphoanhydride bond. A structural rationale is provided for the ability of Arabidopsis inositol tris/tetrakisphosphate kinase 1 to discriminate between symmetric and enantiomeric substrates in the production of diverse symmetric and asymmetric myo-inositol phosphate and diphospho-myo-inositol phosphate (inositol pyrophosphate) products. Simple tools are applied to chromatographic resolution and detection of known and novel diphosphoinositol phosphates without resort to radiolabeling approaches. It is shown that inositol tris/tetrakisphosphate kinase 1 and inositol pentakisphosphate 2-kinase comprise a reversible metabolic cassette converting Ins(3,4,5,6)P4 into 5-InsP7 and back in a nucleotide-dependent manner. Thus, inositol tris/tetrakisphosphate kinase 1 is a nexus of bioenergetics status and inositol polyphosphate/diphosphoinositol phosphate metabolism. As such, it commands a role in plants that evolution has assigned to a different class of enzyme in mammalian cells. The findings and the methods described will enable a full appraisal of the role of diphosphoinositol phosphates in plants and particularly the relative contribution of reversible inositol phosphate hydroxykinase and inositol phosphate phosphokinase activities to plant physiology.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfatos de Inositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Trifosfato de Adenosina/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica/métodos , Fosfatos de Inositol/análise , Mesilatos/química , Mutação , Radioisótopos de Fósforo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Especificidade por SubstratoRESUMO
Many subsurface microorganisms couple their metabolism to the reduction or oxidation of extracellular substrates. For example, anaerobic mineral-respiring bacteria can use external metal oxides as terminal electron acceptors during respiration. Porin-cytochrome complexes facilitate the movement of electrons generated through intracellular catabolic processes across the bacterial outer membrane to these terminal electron acceptors. In the mineral-reducing model bacterium Shewanella oneidensis MR-1, this complex is composed of two decaheme cytochromes (MtrA and MtrC) and an outer-membrane ß-barrel (MtrB). However, the structures and mechanisms by which porin-cytochrome complexes transfer electrons are unknown. Here, we used small-angle neutron scattering (SANS) to study the molecular structure of the transmembrane complexes MtrAB and MtrCAB. Ab initio modeling of the scattering data yielded a molecular envelope with dimensions of â¼105 × 60 × 35 Å for MtrAB and â¼170 × 60 × 45 Å for MtrCAB. The shapes of these molecular envelopes suggested that MtrC interacts with the surface of MtrAB, extending â¼70 Å from the membrane surface and allowing the terminal hemes to interact with both MtrAB and an extracellular acceptor. The data also reveal that MtrA fully extends through the length of MtrB, with â¼30 Å being exposed into the periplasm. Proteoliposome models containing membrane-associated MtrCAB and internalized small tetraheme cytochrome (STC) indicate that MtrCAB could reduce Fe(III) citrate with STC as an electron donor, disclosing a direct interaction between MtrCAB and STC. Taken together, both structural and proteoliposome experiments support porin-cytochrome-mediated electron transfer via periplasmic cytochromes such as STC.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Membrana Celular/metabolismo , Grupo dos Citocromos c/química , Elétrons , Metais/química , Periplasma/metabolismo , Shewanella/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Respiração Celular , Cristalografia por Raios X , Grupo dos Citocromos c/metabolismo , Transporte de Elétrons , Metais/metabolismo , OxirreduçãoRESUMO
Of the many biophysical techniques now being brought to bear on studies of membranes, electron paramagnetic resonance (EPR) of nitroxide spin probes was the first to provide information about both mobility and ordering in lipid membranes. Here, we report the first prediction of variable temperature EPR spectra of model lipid bilayers in the presence and absence of cholesterol from the results of large scale fully atomistic molecular dynamics (MD) simulations. Three types of structurally different spin probes were employed in order to study different parts of the bilayer. Our results demonstrate very good agreement with experiment and thus confirm the accuracy of the latest lipid force fields. The atomic resolution of the simulations allows the interpretation of the molecular motions and interactions in terms of their impact on the sensitive EPR line shapes. Direct versus indirect effects of cholesterol on the dynamics of spin probes are analysed. Given the complexity of structural organisation in lipid bilayers, the advantage of using a combined MD-EPR simulation approach is two-fold. Firstly, prediction of EPR line shapes directly from MD trajectories of actual phospholipid structures allows unambiguous interpretation of EPR spectra of biological membranes in terms of complex motions. Secondly, such an approach provides an ultimate test bed for the up-to-date MD simulation models employed in the studies of biological membranes, an area that currently attracts great attention.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Bicamadas Lipídicas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Colesterol/química , Simulação de Dinâmica Molecular , Óxido Nítrico/química , Marcadores de Spin , TemperaturaRESUMO
The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar-assisted chemical synthesis. In Shewanella oneidensis MR-1 (MR-1), trans-outer-membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer-membrane-spanning porinâ cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosinâ Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR-1. We show photoreduction of MtrC and OmcA adsorbed on RuII -dye-sensitized TiO2 nanoparticles and that these protein-coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer-membrane-associated cytochromes of MR-1 for solar-driven microbial synthesis in natural and engineered bacteria.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Corantes/química , Grupo dos Citocromos c/metabolismo , Titânio/química , Catálise , Transporte de Elétrons , Amarelo de Eosina-(YS)/química , Compostos Férricos/química , Mononucleotídeo de Flavina/química , Luz , Nanopartículas Metálicas/química , Oxirredução , Fármacos Fotossensibilizantes/química , ShewanellaRESUMO
The mineral-respiring bacterium Shewanella oneidensis uses a protein complex, MtrCAB, composed of two decaheme cytochromes, MtrC and MtrA, brought together inside a transmembrane porin, MtrB, to transport electrons across the outer membrane to a variety of mineral-based electron acceptors. A proteoliposome system containing a pool of internalized electron carriers was used to investigate how the topology of the MtrCAB complex relates to its ability to transport electrons across a lipid bilayer to externally located Fe(III) oxides. With MtrA facing the interior and MtrC exposed on the outer surface of the phospholipid bilayer, the established in vivo orientation, electron transfer from the interior electron carrier pool through MtrCAB to solid-phase Fe(III) oxides was demonstrated. The rates were 10(3) times higher than those reported for reduction of goethite, hematite, and lepidocrocite by S. oneidensis, and the order of the reaction rates was consistent with those observed in S. oneidensis cultures. In contrast, established rates for single turnover reactions between purified MtrC and Fe(III) oxides were 10(3) times lower. By providing a continuous flow of electrons, the proteoliposome experiments demonstrate that conduction through MtrCAB directly to Fe(III) oxides is sufficient to support in vivo, anaerobic, solid-phase iron respiration.
Assuntos
Citocromos/metabolismo , Transporte de Elétrons/fisiologia , Compostos Férricos/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Shewanella/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/química , Grupo dos Citocromos c/metabolismo , Immunoblotting , Anotação de Sequência Molecular , Dados de Sequência MolecularRESUMO
Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular intercytochrome electron exchange along "nanowire" appendages. We present a 3.2-Å crystal structure of one of these decaheme cytochromes, MtrF, that allows the spatial organization of the 10 hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65-Å octaheme chain transects the length of the protein and is bisected by a planar 45-Å tetraheme chain that connects two extended Greek key split ß-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g., minerals), soluble substrates (e.g., flavins), and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Grupo dos Citocromos c/química , Citocromos/química , Heme/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Citocromos/genética , Citocromos/metabolismo , Dissulfetos/química , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Mononucleotídeo de Flavina/farmacologia , Heme/metabolismo , Ferro/química , Ferro/metabolismo , Ferro/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução/efeitos dos fármacos , Potenciometria , Ligação Proteica , Estrutura Terciária de Proteína , Shewanella/genética , Shewanella/metabolismoRESUMO
Many species of bacteria can couple anaerobic growth to the respiratory reduction of insoluble minerals containing Fe(III) or Mn(III/IV). It has been suggested that in Shewanella species electrons cross the outer membrane to extracellular substrates via 'porin-cytochrome' electron transport modules. The molecular structure of an outer-membrane extracellular-facing deca-haem terminus for such a module has recently been resolved. It is debated how, once outside the cells, electrons are transferred from outer-membrane cytochromes to insoluble electron sinks. This may occur directly or by assemblies of cytochromes, perhaps functioning as 'nanowires', or via electron shuttles. Here we review recent work in this field and explore whether it allows for unification of the electron transport mechanisms supporting extracellular mineral respiration in Shewanella that may extend into other genera of Gram-negative bacteria.
Assuntos
Citocromos/metabolismo , Transporte de Elétrons , Minerais/metabolismo , Porinas/metabolismo , Shewanella/fisiologia , Anaerobiose , Modelos Biológicos , Oxirredução , Shewanella/crescimento & desenvolvimento , Shewanella/metabolismoRESUMO
Many bacteria of the genus Shewanella are facultative anaerobes able to reduce a broad range of soluble and insoluble substrates, including Fe(III) mineral oxides. Under anoxic conditions, the bacterium Shewanella oneidensis MR-1 uses a porin-cytochrome complex (Mtr) to mediate extracellular electron transfer (EET) across the outer membrane to extracellular substrates. However, it is unclear how EET prevents generating harmful reactive oxygen species (ROS) when exposed to oxic environments. The Mtr complex is expressed under anoxic and oxygen-limited conditions and contains an extracellular MtrC subunit. This has a conserved CX8C motif that inhibits aerobic growth when removed. This inhibition is caused by an increase in ROS that kills the majority of S. oneidensis cells in culture. To better understand this effect, soluble MtrC isoforms with modified CX8C were isolated. These isoforms produced increased concentrations of H2O2 in the presence of flavin mononucleotide (FMN) and greatly increased the affinity between MtrC and FMN. X-ray crystallography revealed that the molecular structure of MtrC isoforms was largely unchanged, while small-angle X-ray scattering suggested that a change in flexibility was responsible for controlling FMN binding. Together, these results reveal that FMN reduction in S. oneidensis MR-1 is controlled by the redox-active disulfide on the cytochrome surface. In the presence of oxygen, the disulfide forms, lowering the affinity for FMN and decreasing the rate of peroxide formation. This cysteine pair consequently allows the cell to respond to changes in oxygen level and survive in a rapidly transitioning environment. IMPORTANCE Bacteria that live at the oxic/anoxic interface have to rapidly adapt to changes in oxygen levels within their environment. The facultative anaerobe Shewanella oneidensis MR-1 can use EET to respire in the absence of oxygen, but on exposure to oxygen, EET could directly reduce extracellular oxygen and generate harmful reactive oxygen species that damage the bacterium. By modifying an extracellular cytochrome called MtrC, we show how preventing a redox-active disulfide from forming causes the production of cytotoxic concentrations of peroxide. The disulfide affects the affinity of MtrC for the redox-active flavin mononucleotide, which is part of the EET pathway. Our results demonstrate how a cysteine pair exposed on the surface controls the path of electron transfer, allowing facultative anaerobic bacteria to rapidly adapt to changes in oxygen concentration at the oxic/anoxic interface.
Assuntos
Cisteína , Shewanella , Espécies Reativas de Oxigênio/metabolismo , Cisteína/metabolismo , Compostos Férricos/metabolismo , Mononucleotídeo de Flavina/metabolismo , Peróxido de Hidrogênio/metabolismo , Oxirredução , Citocromos/metabolismo , Transporte de Elétrons , Shewanella/genética , Shewanella/metabolismo , Flavinas/metabolismo , Oxigênio/metabolismo , Dissulfetos/metabolismoRESUMO
The mineral-respiring bacterium Shewanella oneidensis uses a protein complex, MtrCAB, composed of two decahaem cytochromes brought together inside a transmembrane porin to transport electrons across the outer membrane to a variety of mineral-based electron acceptors. A proteoliposome system has been developed that contains Methyl Viologen as an internalized electron carrier and valinomycin as a membrane-associated cation exchanger. These proteoliposomes can be used as a model system to investigate MtrCAB function.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Lipossomos/química , Shewanella/metabolismo , Proteínas da Membrana Bacteriana Externa/fisiologia , Transporte de Elétrons , Modelos Biológicos , Oxirredução , Paraquat/química , ValinomicinaRESUMO
Many species of the bacterial Shewanella genus are notable for their ability to respire in anoxic environments utilizing insoluble minerals of Fe(III) and Mn(IV) as extracellular electron acceptors. In Shewanella oneidensis, the process is dependent on the decahaem electron-transport proteins that lie at the extracellular face of the outer membrane where they can contact the insoluble mineral substrates. These extracellular proteins are charged with electrons provided by an inter-membrane electron-transfer pathway that links the extracellular face of the outer membrane with the inner cytoplasmic membrane and thereby intracellular electron sources. In the present paper, we consider the common structural features of two of these outer-membrane decahaem cytochromes, MtrC and MtrF, and bring this together with biochemical, spectroscopic and voltammetric data to identify common and distinct properties of these prototypical members of different clades of the outer-membrane decahaem cytochrome superfamily.
Assuntos
Espaço Extracelular/metabolismo , Ferro/metabolismo , Minerais/metabolismo , Shewanella/metabolismo , Aerobiose , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dados de Sequência Molecular , Oxirredução , Shewanella/genéticaRESUMO
Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic and anaerobic metabolism. In the absence of O(2), FNR binds a [4Fe-4S](2+) cluster (ligated by Cys-20, 23, 29, 122) promoting the formation of a transcriptionally active dimer. In the presence of O(2), FNR is converted into a monomeric, non-DNA-binding form containing a [2Fe-2S](2+) cluster. The reaction of the [4Fe-4S](2+) cluster with O(2) has been shown to proceed via a 2-step process, an O(2)-dependent 1-electron oxidation to yield a [3Fe-4S](+) intermediate with release of 1 Fe(2+) ion, followed by spontaneous rearrangement to the [2Fe-2S](2+) form with release of 1 Fe(3+) and 2 S(2-) ions. Here, we show that replacement of Ser-24 by Arg, His, Phe, Trp, or Tyr enhances aerobic activity of FNR in vivo. The FNR-S24F protein incorporates a [4Fe-4S](2+) cluster with spectroscopic properties similar to those of FNR. However, the substitution enhances the stability of the [4Fe-4S](2+) cluster in the presence of O(2). Kinetic analysis shows that both steps 1 and 2 are slower for FNR-S24F than for FNR. A molecular model suggests that step 1 of the FNR-S24F iron-sulfur cluster reaction with O(2) is inhibited by shielding of the iron ligand Cys-23, suggesting that Cys-23 or the cluster iron bound to it is a primary site of O(2) interaction. These data lead to a simple model of the FNR switch with physiological implications for the ability of FNR proteins to operate over different ranges of in vivo O(2) concentrations.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Oxigênio/metabolismo , Serina/metabolismo , Fatores de Transcrição/metabolismo , Aerobiose , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Escherichia coli/química , Proteínas Ferro-Enxofre/química , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Fenilalanina/genética , Estabilidade Proteica , Espectrofotometria Ultravioleta , Relação Estrutura-Atividade , Fatores de Transcrição/químicaRESUMO
The Tat system is used to transport folded proteins across the cytoplasmic membrane in bacteria and archaea and across the thylakoid membrane of plant chloroplasts. Multimers of the integral membrane TatA protein are thought to form the protein-conducting element of the Tat pathway. Nitroxide radicals were introduced at selected positions within the transmembrane helix of Escherichia coli TatA and used to probe the structure of detergent-solubilized TatA complexes by EPR spectroscopy. A comparison of spin label mobilities allowed classification of individual residues as buried within the TatA complex or exposed at the surface and suggested that residues Ile(12) and Val(14) are involved in interactions between helices. Analysis of inter-spin distances suggested that the transmembrane helices of TatA subunits are arranged as a single-walled ring containing a contact interface between Ile(12) on one subunit and Val(14) on an adjacent subunit. Experiments in which labeled and unlabeled TatA samples were mixed demonstrate that TatA subunits are exchanged between TatA complexes. This observation is consistent with the TatA dynamic polymerization model for the mechanism of Tat transport.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Subunidades Proteicas/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Plasmídeos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Marcadores de SpinRESUMO
Heme, a physiologically crucial form of iron, is a cofactor for a very wide range of proteins and enzymes. These include DNA regulatory proteins in which heme is a sensor to which an analyte molecule binds, effecting a change in the DNA binding affinity of the regulator. Given that heme, and more generally iron, must be carefully regulated, it is surprising that there are no examples yet in bacteria in which heme itself is sensed directly by a reversibly binding DNA regulatory protein. Here we show that the Rhizobium leguminosarum global iron regulatory protein Irr, which has many homologues within the alpha-proteobacteria and is a member of the Fur superfamily, binds heme, resulting in a dramatic decrease in affinity between the protein and its cognate, regulatory DNA operator sequence. Spectroscopic studies of wild-type and mutant Irr showed that the principal (but not only) heme-binding site is at a conserved HXH motif, whose substitution led to loss of DNA binding in vitro and of regulatory function in vivo. The R. leguminosarum Irr behaves very differently to the Irr of Bradyrhizobium japonicum, which is rapidly degraded in vivo by an unknown mechanism in conditions of elevated iron or heme, but whose DNA binding affinity in vitro does not respond to heme.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Heme/metabolismo , Regiões Operadoras Genéticas/fisiologia , Rhizobium leguminosarum/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Bradyrhizobium/genética , Bradyrhizobium/metabolismo , DNA Bacteriano/genética , Heme/genética , Mutação , Ligação Proteica/fisiologia , Rhizobium leguminosarum/genética , Especificidade da Espécie , Fatores de Transcrição/genéticaRESUMO
Src homology 2 domain-containing inositol phosphate phosphatase 2 (SHIP2) is one of the 10 human inositol phosphate 5-phosphatases. One of its physiological functions is dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate, PtdIns(3,4,5)P3. It is therefore a therapeutic target for pathophysiologies dependent on PtdIns(3,4,5)P3 and PtdIns(3,4)P2. Therapeutic interventions are limited by the dearth of crystallographic data describing ligand/inhibitor binding. An active site-directed fluorescent probe facilitated screening of compound libraries for SHIP2 ligands. With two additional orthogonal assays, several ligands including galloflavin were identified as low micromolar Ki inhibitors. One ligand, an oxo-linked ethylene-bridged dimer of benzene 1,2,4-trisphosphate, was shown to be an uncompetitive inhibitor that binds to a regulatory site on the catalytic domain. We posit that binding of ligands to this site restrains L4 loop motions that are key to interdomain communications that accompany high catalytic activity with phosphoinositide substrate. This site may, therefore, be a future druggable target for medicinal chemistry.
Assuntos
Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Fosfatos de Inositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Sítio Alostérico , Sequência de Aminoácidos , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Ligantes , Camundongos , Simulação de Acoplamento Molecular , Células NIH 3T3 , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Ligação ProteicaRESUMO
A nitroxide spin label (SL) has been used to probe the electron spin relaxation times and the magnetic states of the oxygen-binding heme-copper dinuclear site in Escherichia coli cytochrome bo(3), a quinol oxidase (QO), in different oxidation states. The spin lattice relaxation times, T(1), of the SL are enhanced by the paramagnetic metal sites in QO and hence show a strong dependence on the oxidation state of the latter. A new, general form of equations and a computer simulation program have been developed for the calculation of relaxation enhancement by an arbitrary fast relaxing spin system of S ≥ 1/2. This has allowed us to obtain an accurate estimate of the transverse relaxation time, T (2), of the dinuclear coupled pair Fe(III)-Cu(B)(II) in the oxidized form of QO that is too short to measure directly. In the case of the F' state, the relaxation properties of the heme-copper center have been shown to be consistent with a ferryl [Fe(IV)=O] heme and Cu(B)(II) coupled by approximately 1.5-3 cm(-1) to a radical. The magnitude suggests that the coupling arises from a radical form of the covalently linked tyrosine-histidine ligand to Cu(II) with unpaired spin density primarily on the tyrosine component. This work demonstrates that nitroxide SLs are potentially valuable tools to probe both the relaxation and the magnetic properties of multinuclear high-spin paramagnetic active sites in proteins that are otherwise not accessible from direct EPR measurements.
Assuntos
Cobre/química , Citocromos/química , Proteínas de Escherichia coli/química , Heme/química , Magnetismo , Óxidos de Nitrogênio/química , Marcadores de Spin , Sítios de Ligação , Cobre/metabolismo , Grupo dos Citocromos b , Citocromos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Heme/metabolismo , Ferro/química , Ferro/metabolismo , Modelos Moleculares , Óxidos de Nitrogênio/metabolismo , OxirreduçãoRESUMO
SHIP2 (SH2-domain containing inositol 5-phosphatase type 2) is a canonical 5-phosphatase, which, through its catalytic action on PtdInsP3, regulates the PI3K/Akt pathway and metabolic action of insulin. It is a drug target, but there is limited evidence of inhibition of SHIP2 by small molecules in the literature. With the goal to investigate inhibition, we report a homologous family of synthetic, chromophoric benzene phosphate substrates of SHIP2 that display the headgroup regiochemical hallmarks of the physiological inositide substrates that have proved difficult to crystallize with 5-phosphatases. Using time-dependent density functional theory (TD-DFT), we explore the intrinsic fluorescence of these novel substrates and show how fluorescence can be used to assay enzyme activity. The TD-DFT approach promises to inform rational design of enhanced active site probes for the broadest family of inositide-binding/metabolizing proteins, while maintaining the regiochemical properties of bona fide inositide substrates.
RESUMO
The metabolic flexibility of bacteria is key to their ability to survive and thrive in a wide range of environments. Optimal switching from one metabolic pathway to another is a key requirement for this flexibility. Respiration is a good example: many bacteria utilize O(2) as the terminal electron acceptor, but can switch to a range of other acceptors, such as nitrate, when O(2) becomes limiting. Sensing environmental levels of O(2) is the key step in switching from aerobic to anaerobic respiration. In Escherichia coli, the fumarate and nitrate reduction transcriptional regulator (FNR) controls this switch. Under O(2)-limiting conditions, FNR binds a [4Fe-4S](2+) cluster, generating a transcriptionally active dimeric form. Exposure to O(2) results in conversion of the cluster into a [2Fe-2S](2+) form, leading to dissociation of the protein into inactive monomers. The mechanism of cluster conversion, together with the nature of the reaction products, is of considerable current interest, and a near-complete description of the process has now emerged. The [4Fe-4S](2+) into [2Fe-2S](2+) cluster conversion proceeds via a two-step mechanism. In step 1, a one-electron oxidation of the cluster takes place, resulting in the release of a Fe(2+) ion, the formation of an intermediate [3Fe-4S](1+) cluster, together with the generation of a superoxide anion. In step 2, the intermediate [3Fe-4S](1+) cluster rearranges spontaneously to form the [2Fe-2S](2+) cluster, releasing two sulfide ions and an Fe(3+) ion in the process. The one-electron activation of the cluster, coupled to catalytic recycling of the superoxide anion back to oxygen via superoxide dismutase and catalase, provides a novel means of amplifying the sensitivity of [4Fe-4S](2+) FNR to its signal molecule.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Escherichia coli/química , Proteínas Ferro-Enxofre/química , Oxirredução , Oxigênio/metabolismo , Estrutura Secundária de Proteína , Transcrição GênicaRESUMO
Certain bacterial species have a natural ability to exchange electrons with extracellular redox partners. This behavior allows coupling of catalytic transformations inside bacteria to complementary redox transformations of catalysts and electrodes outside the cell. Electricity generation can be coupled to waste-water remediation. Industrially relevant oxidation reactions can proceed exclusively when electrons are released to anodes. Reduced products such as fuels can be generated when electrons are provided from (photo)cathodes. Rational development of these opportunities and inspiration for novel technologies is underpinned by resolution at the molecular level of pathways supporting electron exchange across bacterial cell envelopes. This chapter describes methods for purification, engineering, and in vitro characterization of proteins providing the primary route for electron transport across the outer membrane lipid bilayer of Shewanella oneidensis MR-1, a well-described electrogenic bacterium and chassis organism for related biotechnologies.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Transporte de Elétrons/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Oxirredução , Shewanella/enzimologiaRESUMO
The bioenergetics of anaerobic metabolism frequently relies on redox loops performed by membrane complexes with substrate- and quinone-binding sites on opposite sides of the membrane. However, in sulfate respiration (a key process in the biogeochemical sulfur cycle), the substrate- and quinone-binding sites of the QrcABCD complex are periplasmic, and their role in energy conservation has not been elucidated. Here we show that the QrcABCD complex of Desulfovibrio vulgaris is electrogenic, as protons and electrons required for quinone reduction are extracted from opposite sides of the membrane, with a H+/e- ratio of 1. Although the complex does not act as a H+-pump, QrcD may include a conserved proton channel leading from the N-side to the P-side menaquinone pocket. Our work provides evidence of how energy is conserved during dissimilatory sulfate reduction, and suggests mechanisms behind the functions of related bacterial respiratory complexes in other bioenergetic contexts.