RESUMO
It had been proposed that a loop, typically containing 26 or 27 amino acids, which is only present in monomeric, ferredoxin-dependent, "plant-type" glutamate synthases and is absent from the catalytic α-subunits of both NADPH-dependent, heterodimeric glutamate synthases found in non-photosynthetic bacteria and NADH-dependent heterodimeric cyanobacterial glutamate synthases, plays a key role in productive binding of ferredoxin to the plant-type enzymes. Site-directed mutagenesis has been used to delete the entire 27 amino acid-long loop in the ferredoxin-dependent glutamate synthase from the cyanobacterium Synechocystis sp. PCC 6803. The specific activity of the resulting loopless variant of this glutamate synthase, when reduced ferredoxin serves as the electron donor, is actually higher than that of the wild-type enzyme, suggesting that this loop is not absolutely essential for efficient electron transfer from reduced ferredoxin to the enzyme. These results are consistent with the results of an in-silico study that suggests that the loop is unlikely to interact directly with ferredoxin in the energetically most favorable model of a 1:1 complex of ferredoxin with the wild-type enzyme.
Assuntos
Aminoácido Oxirredutases/metabolismo , Ferredoxinas/metabolismo , Ácido Glutâmico/biossíntese , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/genética , Sequência de Aminoácidos , Catálise , Simulação por Computador , Cinética , Redes e Vias Metabólicas , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Synechocystis/enzimologia , Synechocystis/genéticaRESUMO
The roles of four conserved basic amino acids in the reaction catalyzed by the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 have been investigated using site-directed mutagenesis in combination with measurements of steady-state kinetics, substrate-binding affinities, and spectroscopic properties of the enzyme's two prosthetic groups. Replacement of either Lys58 or Arg70 by glutamine leads to a complete loss of activity, both with the physiological electron donor, reduced ferredoxin, and with a nonphysiological electron donor, reduced methyl viologen. More conservative, charge-maintaining K58R and R70K variants were also completely inactive. Replacement of Lys130 by glutamine produced a variant that retained 26% of the wild-type activity with methyl viologen as the electron donor and 22% of the wild-type activity with ferredoxin as the electron donor, while replacement by arginine produces a variant that retains a significantly higher percentage of the wild-type activity with both electron donors. In contrast, replacement of Arg146 by glutamine had minimal effect on the activity of the enzyme. These results, along with substrate-binding and spectroscopic measurements, are discussed in terms of an in silico structural model for the enzyme.
Assuntos
Aminoácidos Básicos/química , Ferredoxinas/química , Nitrato Redutase/química , Synechococcus/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sequência Conservada , Glutamina/química , Glutamina/genética , Dados de Sequência Molecular , Nitrato Redutase/genética , Especificidade por Substrato/genética , Synechococcus/genéticaRESUMO
The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E(m)) value of -165mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E(m) value of -220mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.
Assuntos
Arseniato Redutases/química , Proteínas de Bactérias/química , Glutarredoxinas/química , Synechocystis/enzimologia , Arseniato Redutases/genética , Arseniatos/metabolismo , Biocatálise , Clonagem Molecular , Cisteína/química , Glutationa/química , Dados de Sequência Molecular , Oxirredução , Homologia de Sequência de AminoácidosRESUMO
Ferredoxin (Fd) is the major iron-containing protein in photosynthetic organisms and is central to reductive metabolism in the chloroplast. The Chlamydomonas reinhardtii genome encodes six plant type [Fe2S2] ferredoxins, products of PETF, FDX2-FDX6. We performed the functional analysis of these ferredoxins by localizing Fd, Fdx2, Fdx3, and Fdx6 to the chloroplast by using isoform-specific antibodies and monitoring the pattern of gene expression by iron and copper nutrition, nitrogen source, and hydrogen peroxide stress. In addition, we also measured the midpoint redox potentials of Fd and Fdx2 and determined the kinetic parameters of their reactions with several ferredoxin-interacting proteins, namely nitrite reductase, Fd:NADP+ oxidoreductase, and Fd:thioredoxin reductase. We found that each of the FDX genes is differently regulated in response to changes in nutrient supply. Moreover, we show that Fdx2 (Em = -321 mV), whose expression is regulated by nitrate, is a more efficient electron donor to nitrite reductase relative to Fd. Overall, the results suggest that each ferredoxin isoform has substrate specificity and that the presence of multiple ferredoxin isoforms allows for the allocation of reducing power to specific metabolic pathways in the chloroplast under various growth conditions.
Assuntos
Proteínas de Algas/biossíntese , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Ferredoxinas/biossíntese , Regulação da Expressão Gênica/fisiologia , Proteínas de Protozoários/biossíntese , Proteínas de Algas/genética , Animais , Sequência de Bases , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Cloroplastos/genética , Cobre/metabolismo , Cobre/farmacologia , Ferredoxinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma de Cloroplastos/fisiologia , Peróxido de Hidrogênio/farmacocinética , Ferro/metabolismo , Ferro/farmacologia , Dados de Sequência Molecular , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Protozoários/genéticaRESUMO
The ferredoxin-dependent nitrite reductase from the green alga Chlamydomonas reinhardtii has been cloned, expressed in Escherichia coli as a His-tagged recombinant protein, and purified to homogeneity. The spectra, kinetic properties and substrate-binding parameters of the C. reinhardtii enzyme are quite similar to those of the ferredoxin-dependent spinach chloroplast nitrite reductase. Computer modeling, based on the published structure of spinach nitrite reductase, predicts that the structure of C. reinhardtii nitrite reductase will be similar to that of the spinach enzyme. Chemical modification studies and the ionic-strength dependence of the enzyme's ability to interact with ferredoxin are consistent with the involvement of arginine and lysine residues on C. reinhardtii nitrite reductase in electrostatically-stabilized binding to ferredoxin. The C. reinhardtii enzyme has been used to demonstrate that hydroxylamine can serve as an electron-accepting substrate for the enzyme and that the product of hydroxylamine reduction is ammonia, providing the first experimental evidence for the hypothesis that hydroxylamine, bound to the enzyme, can serve as a late intermediate during the reduction of nitrite to ammonia catalyzed by the enzyme.
Assuntos
Amônia/metabolismo , Chlamydomonas reinhardtii/enzimologia , Ferredoxina-Nitrito Redutase/metabolismo , Hidroxilamina/metabolismo , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Ferredoxina-Nitrito Redutase/química , Ferredoxinas/metabolismo , Modelos Moleculares , Nitritos/metabolismo , Concentração Osmolar , Oxirredução , Estrutura Secundária de Proteína , Proteínas Recombinantes/metabolismo , Spinacia oleracea/enzimologiaRESUMO
Nitrite reductase, which reduces nitrite to ammonium in a six-electron reaction, was characterized through kinetic analysis of an electron transfer cascade involving photoexcited Photosystem I and ferredoxin. This cascade was studied at physiological pH by flash-absorption spectroscopy. Two different forms of the enzyme were studied: one isolated from spinach leaf and one histidine-tagged recombinant form. When the enzyme is oxidized in the absence of nitrite, single-enzyme reduction leads mostly to siroheme reduction with the leaf enzyme, whereas the siroheme and the [4Fe-4S] cluster are both reduced in equivalent amounts in the recombinant enzyme. When combined with the results of deazaflavin/EDTA photoreduction experiments, these data support a 50 mV negative shift of the siroheme midpoint potential in the recombinant enzyme. Despite this difference, the two forms of the enzyme exhibit similar values for the rate constant of single reduction by reduced ferredoxin (1200 s(-1)) and for k(cat) (420-450 electrons per second and per nitrite reductase). When nitrite reductase is initially pre-reduced to the state ferrous siroheme-NO(*), the fast kinetics of reduction by ferredoxin and the thermodynamics of ferredoxin binding are equivalent to those observed with oxidized nitrite reductase without nitrite. Spectral and kinetic analyses of single reduction of the recombinant enzyme in the ferrous siroheme-NO(*) state by photoreduced ferredoxin reveal that this process leads to reduction of the [4Fe-4S] cluster with little, if any, NO(*) reduction. These data show that the enzyme must wait for the next reduction step before NO(*) undergoes substantial reduction.
Assuntos
Nitrito Redutases/química , Proteínas de Plantas/química , Sítios de Ligação , Catálise , Transporte de Elétrons , Cinética , Nitrito Redutases/metabolismo , Nitritos/química , Nitritos/metabolismo , Oxirredução , Proteínas de Plantas/metabolismo , Synechocystis/enzimologia , Synechocystis/metabolismo , TermodinâmicaRESUMO
In oxygenic photosynthetic cells, carbon metabolism is regulated by a light-dependent redox signaling pathway through which the light signal is transmitted in the form of electrons via a redox chain comprising ferredoxin (Fd), ferredoxin:thioredoxin reductase (FTR), and thioredoxin (Trx). Trx affects the activity of a variety of enzymes via dithiol oxidation and reduction reactions. FTR reduces an intramolecular disulfide bridge of Trx, and Trx reduction involves a transient cross-link with FTR. NMR spectroscopy was used to investigate the interaction of Fd, FTR, and an m-type Trx. NMR titration experiments indicate that FTR uses distinct sites to bind Fd and Trx simultaneously to form a noncovalent ternary complex. The orientation of Trx-m relative to FTR was determined from the intermolecular paramagnetic broadening caused by the [4Fe-4S] cluster of FTR. Two models of the noncovalent binary complex of FTR/Trx-m based on the paramagnetic distance restraints were obtained. The models suggest that either a modest or major rotational movement of Trx must take place when the noncovalent binary complex proceeds to the covalent complex. This study demonstrates the complementarity of paramagnetic NMR and X-ray diffraction of crystals in the elucidation of dynamics in a transient protein complex.
Assuntos
Ferredoxinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Oxirredutases/metabolismo , Tiorredoxinas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Ferredoxinas/química , Proteínas Ferro-Enxofre/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Movimento , Oxirredutases/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ligação Proteica , Soluções , Spinacia oleracea , Synechocystis/enzimologia , Tiorredoxinas/químicaRESUMO
Photosynthetic [2Fe-2S] plant-type ferredoxins have a central role in electron transfer between the photosynthetic chain and various metabolic pathways. Several genes are coding for [2Fe2S] ferredoxins in cyanobacteria, with four in the thermophilic cyanobacterium Thermosynechococcus elongatus. The structure and functional properties of the major ferredoxin Fd1 are well known but data on the other ferredoxins are scarce. We report the structural and functional properties of a novel minor type ferredoxin, Fd2 of T. elongatus, homologous to Fed4 from Synechocystis sp. PCC 6803. Remarkably, the midpoint potential of Fd2, Emâ¯=â¯-440â¯mV, is lower than that of Fd1, Emâ¯=â¯-372â¯mV. However, while Fd2 can efficiently react with photosystem I or nitrite reductase, time-resolved spectroscopy shows that Fd2 has a very low capacity to reduce ferredoxin-NADP+ oxidoreductase (FNR). These unique Fd2 properties are discussed in relation with its structure, solved at 1.38â¯Å resolution. The Fd2 structure significantly differs from other known ferredoxins structures in loop 2, N-terminal region, hydrogen bonding networks and surface charge distributions. UV-Vis, EPR, and Mid- and Far-IR data also show that the electronic properties of the [2Fe2S] cluster of Fd2 and its interaction with the protein differ from those of Fd1 both in the oxidized and reduced states. The structural analysis allows to propose that valine in the motif Cys53ValAsnCys56 of Fd2 and the specific orientation of Phe72, explain the electron transfer properties of Fd2. Strikingly, the nature of these residues correlates with different phylogenetic groups of cyanobacterial Fds. With its low redox potential and its discrimination against FNR, Fd2 exhibits a unique capacity to direct efficiently photosynthetic electrons to metabolic pathways not dependent on FNR.
Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Ferredoxinas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cianobactérias/genética , Ferredoxinas/química , Ferredoxinas/genética , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Filogenia , Alinhamento de Sequência , ThermosynechococcusRESUMO
The reduction of ferredoxin-thioredoxin reductase (FTR) by plant-type ferredoxin plays an important role in redox regulation in plants and cyanobacteria. Nuclear magnetic resonance (NMR) was used to map the binding sites on Synechocystis ferredoxin for FTR. A gallium-substituted structural analog of this [2Fe-2S] ferredoxin was obtained by reconstituting the apoprotein in a refolding buffer containing gallium. For the first time, the complete interaction interface of a [2Fe-2S] ferredoxin with a target enzyme has been mapped by NMR chemical shift perturbation with this diamagnetic structural analog.
Assuntos
Ferredoxinas/metabolismo , Gálio/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Oxirredutases/metabolismo , Synechocystis/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Ferredoxinas/química , Proteínas Ferro-Enxofre/química , Modelos Moleculares , Dados de Sequência Molecular , Oxirredutases/química , TitulometriaRESUMO
The 23 amino acid-long extracellular domain of the influenza virus transmembrane protein M2 (M2e) has remained highly conserved since the 1918 pandemic, and is thus considered a good candidate for development of a universal influenza A vaccine. However, M2e is poorly immunogenic. In this study we assessed the potential of increasing immunogenicity of M2e by constructing a nanoscale-designed protein polymer containing the M2e sequence and an elastin-like polypeptide (ELP) nanodomain consisting of alanine and tyrosine guest residues (ELP(A2YA2)24). The ELP nanodomain was included to increase antigen size, and to exploit the inherent thermal inverse phase transition behavior of ELPs to purify the protein polymer. The ELP(A2YA2)24 + M2e nanodomained molecule was recombinantly synthesized. Characterization of its inverse phase transition behavior demonstrated that attachment of M2e to ELP(A2YA2)24 increased its transition temperature compared to ELP(A2YA2)24. Using a dot blot test we determined that M2e conjugated to ELP is recognizable by M2e-specific antibodies, suggesting that the conjugation process does not adversely affect the immunogenic property of M2e. Further, upon vaccinating mice with ELP(A2YA2)24 + M2e it was found that indeed the nanodomained protein enhanced M2e-specific antibodies in mouse serum compared to free M2e peptide and ELP(A2YA2)24. The immune serum could also recognize M2 expressed on influenza virions. Overall, this data suggests the potential of using molecules containing M2e-ELP nano-domains to develop a universal influenza vaccine.
RESUMO
A series of site-directed mutants of the ferredoxin-dependent spinach nitrite reductase has been characterized and several amino acids have been identified that appear to be involved in the interaction of the enzyme with ferredoxin. In a complementary study, binding constants to nitrite reductase and steady-state kinetic parameters of site-directed mutants of ferredoxin were determined in an attempt to identify ferredoxin amino acids involved in the interaction with nitrite reductase. The results have been interpreted in terms of an in-silico docking model for the 1:1 complex of ferredoxin with nitrite reductase.
Assuntos
Sequência Conservada/genética , Ferredoxina-Nitrito Redutase/genética , Ferredoxinas/metabolismo , Mutagênese Sítio-Dirigida , Nitrito Redutases/metabolismo , Sítios de Ligação , Ferredoxina-Nitrito Redutase/metabolismo , Ferredoxinas/genética , Mutação , Nitrito Redutases/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Spinacia oleracea/enzimologiaRESUMO
A system has been developed for expressing a His-tagged form of the ferredoxin-dependent nitrite reductase of spinach in Escherichia coli. The catalytic and spectral properties of the His-tagged, recombinant enzyme are similar, but not identical, to those previously observed for nitrite reductase isolated directly from spinach leaf. A detailed comparison of the spectral, catalytic and fluorescence properties of nitrite reductase variants, in which each of the enzyme's eight tryptophan residues has been replaced using site-directed mutagenesis by either aromatic or non-aromatic amino acids, has been used to examine possible roles for tryptophan residues in the reduction of nitrite to ammonia catalyzed by the enzyme.