29 results on '"Medina, Milagros"'
Search Results
2. Quaternary organization in a bifunctional prokaryotic FAD synthetase: Involvement of an arginine at its adenylyltransferase module on the riboflavin kinase activity
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Serrano, Ana, Sebastián, María, Arilla-Luna, Sonia, Baquedano, Silvia, Pallarés, María Carmen, Lostao, Anabel, Herguedas, Beatriz, Velázquez-Campoy, Adrián, Martínez-Júlvez, Marta, and Medina, Milagros
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- 2015
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- View/download PDF
3. Detection of a quaternary organization into dimer of trimers of Corynebacterium ammoniagenes FAD synthetase at the single-molecule level and at the in cell level
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Marcuello, Carlos, Arilla-Luna, Sonia, Medina, Milagros, and Lostao, Anabel
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- 2013
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4. Coenzyme binding and hydride transfer in Rhodobacter capsulatus ferredoxin/flavodoxin NADP(H) oxidoreductase
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Bortolotti, Ana, Pérez-Dorado, Inmaculada, Goñi, Guillermina, Medina, Milagros, Hermoso, Juan A., Carrillo, Néstor, and Cortez, Néstor
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- 2009
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5. Cyanobacterial electron carrier proteins as electron donors to CYP106A2 from Bacillus megaterium ATCC 13368
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Goñi, Guillermina, Zöllner, Andy, Lisurek, Michael, Velázquez-Campoy, Adrián, Pinto, Sira, Gómez-Moreno, Carlos, Hannemann, Frank, Bernhardt, Rita, and Medina, Milagros
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- 2009
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6. Towards a new interaction enzyme:coenzyme
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Martínez-Júlvez, Marta, Tejero, Jesús, R. Peregrina, José, Nogués, Isabel, Frago, Susana, Gómez-Moreno, Carlos, and Medina, Milagros
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- 2005
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7. Flavin photochemistry in the analysis of electron transfer reactions: role of charged and hydrophobic residues at the carboxyl terminus of ferredoxin–NADP + reductase in the interaction with its substrates
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Faro, Merche, Hurley, John K., Medina, Milagros, Tollin, Gordon, and Gómez-Moreno, Carlos
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- 2002
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8. A hydrogen bond network in the active site of Anabaena ferredoxin-NADP + reductase modulates its catalytic efficiency
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Sánchez Azqueta, Ana, Herguedas, Beatriz, Hurtado Guerrero, R., Hervás Morón, Manuel, Navarro Carruesco, José Antonio, Martínez Júlvez, Marta, Medina, Milagros, Sánchez Azqueta, Ana, Herguedas, Beatriz, Hurtado Guerrero, R., Hervás Morón, Manuel, Navarro Carruesco, José Antonio, Martínez Júlvez, Marta, and Medina, Milagros
- Abstract
Ferredoxin-nicotinamide-adenine dinucleotide phosphate (NADP+) reductase (FNR) catalyses the production of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) in photosynthetic organisms, where its flavin adenine dinucleotide (FAD) cofactor takes two electrons from two reduced ferredoxin (Fd) molecules in two sequential steps, and transfers them to NADP+ in a single hydride transfer (HT) step. Despite the good knowledge of this catalytic machinery, additional roles can still be envisaged for already reported key residues, and new features are added to residues not previously identified as having a particular role in the mechanism. Here, we analyse for the first time the role of Ser59 in Anabaena FNR, a residue suggested by recent theoretical simulations as putatively involved in competent binding of the coenzyme in the active site by cooperating with Ser80. We show that Ser59 indirectly modulates the geometry of the active site, the interaction with substrates and the electronic properties of the isoalloxazine ring, and in consequence the electron transfer (ET) and HT processes. Additionally, we revise the role of Tyr79 and Ser80, previously investigated in homologous enzymes from plants. Our results probe that the active site of FNR is tuned by a H-bond network that involves the side-chains of these residues and that results to critical optimal substrate binding, exchange of electrons and, particularly, competent disposition of the C4n (hydride acceptor/donor) of the nicotinamide moiety of the coenzyme during the reversible HT event.
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- 2014
9. Local estrogenic therapy in patients with ASCUS. Review 2011–2013.
- Author
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Medina, Milagros Martinez, Leira, Jordi, Sanchez, Sonia, Peiro, Ricard, and Rodellar, Laura
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ESTROGEN replacement therapy , *MEDICAL care , *HORMONE therapy , *SQUAMOUS cell carcinoma , *MEDICAL research , *PATIENTS - Published
- 2015
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10. Electron spin echo envelope modulation studies of the semiquinone anion radical of cholesterol oxidase from Brevibacterium sterolicum
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Medina, Milagros, Vrielink, Alice, and Cammack, Richard
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- 1997
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11. Reversible hydrogenase of Anabaena variabilis ATCC 29413: catalytic properties and characterization of redox centres
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Serebryakova, Larissa T., Medina, Milagros, Zorin, Nikolay A., Gogotov, Ivan N., and Cammack, Richard
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- 1996
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12. Understanding the FMN cofactor chemistry within the Anabaena Flavodoxin environment
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Lans, Isaias, Frago, Susana, and Medina, Milagros
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FLAVODOXIN , *ANABAENA , *BIOENERGETICS , *FLAVOPROTEINS , *PHOTOSYNTHESIS , *CHARGE exchange , *METHYL groups - Abstract
Abstract: The chemical versatility of flavin cofactors within the flavoprotein environment allows them to play main roles in the bioenergetics of all type of organisms, particularly in energy transformation processes such as photosynthesis or oxidative phosphorylation. Despite the large diversity of properties shown by flavoproteins and of the biological processes in which they are involved, only two flavin cofactors, FMN and FAD (both derived from the 7,8-dimethyl-10-(1′-D-ribityl)-isoalloxazine), are usually found in these proteins. Using theoretical and experimental approaches we have carried out an evaluation of the effects introduced upon substituting the 7- and/or 8-methyls of the isoalloxazine ring in the chemical and oxido-reduction properties of the different atoms of the ring on free flavins and on the photosynthetic Anabaena Flavodoxin (a flavoprotein that replaces Ferredoxin as electron carrier from Photosystem I to Ferredoxin-NADP+ reductase). In Anabaena Flavodoxin both the protein environment and the redox state contribute to modulate the chemical reactivity of the isoalloxazine ring. Anabaena apoflavodoxin is shown to be designed to stabilise/destabilise each one of the FMN redox states (but not of the analogues produced upon substitution of the 7- and/or 8-methyls groups) in the adequate proportions to provide Flavodoxin with the particular properties required for the functions in which it is involved in vivo. The 7- and/or 8-methyl groups of the ixoalloxazine can be discarded as the gate for electrons exchange in Anabaena Fld, but a key role in this process is envisaged for the C6 atom of the flavin and the backbone atoms of Asn58. [Copyright &y& Elsevier]
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- 2012
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13. The Puzzle of Ligand Binding to Corynebacterium ammonia genes FAD Synthetase.
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Frago, Susana, VeIázquez-Campoy, Adrián, and Medina, Milagros
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CORYNEBACTERIUM , *LIGAND binding (Biochemistry) , *VITAMIN B2 , *PHOSPHORYLATION , *LIGASES , *FLAVINS , *ADENINE nucleotides , *BIOCHEMISTRY - Abstract
In bacteria, riboflavin phosphorylation and subsequent conversion of FMN into FAD are carried out by FAD synthetase, a single bifunctional enzyme. Both reactions require ATP and Mg2+. The N-terminal domain of FAD synthetase appears to be responsible for the adenylyltransferase activity, whereas the C-terminal domain would be in charge of the kinase activity. Binding to Corynebacterium ammoniagenes FAD synthetase of its products and substrates, as well as of several analogues, is analyzed. Binding parameters for adenine nucleotides to each one of the two adenine nucleotide sites are reported. In addition, it is demonstrated for the first time that the enzyme presents two independent flavin sites, each one related with one of the enzymatic activities. The binding parameters of flavins to these sites are also provided. The presence of Mg2+ and of both adenine nucleotides and flavins cooperatively modulates the interaction parameters for the other ligands. Our data also suggest that during its double catalytic cycle FAD synthetase must suffer conformational changes induced byadenine nucleotide-Mg2+ or flavin binding. They might include not only rearrangement of the different protein loops but also alternative conformations between domains. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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14. Flavin photochemistry in the analysis of electron transfer reactions: role of charged and hydrophobic residues at the carboxyl terminus of ferredoxin–NADP+ reductase in the interaction with its substrates
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Faro, Merche, Hurley, John K., Medina, Milagros, Tollin, Gordon, and Gómez-Moreno, Carlos
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CHARGE exchange , *PHOTOSYNTHESIS , *PHOTOCHEMISTRY - Abstract
The enzyme Ferredoxin-NADP+ reductase participates in the reductive side of the photosynthetic chain transferring electrons from reduced Ferredoxin (Fd) (or Flavodoxin (Fld)) to NADP+, a process that yields NADPH that can be used in many biosynthetic dark reactions. The involvement of specific amino acids in the interaction between the two proteins has been studied using site-directed mutagenesis. In the present study, the participation of charged (H299), polar (T302) or hydrophobic (V300) amino acid residues that are in the NADP+-binding domain of the reductase have been examined by analyzing its C-terminal region, which is located close to the active site. Stopped-flow and laser flash photolysis results of the reaction in which these mutant proteins participate show very little differences with respect to the wild-type protein. These results suggest that the NADPH-binding domain of the reductase has little effect on the processes of recognition and electron transfer to (and from) Fd or Fld, according to the recently reported crystallographic structure of the FNR/Fd complex [Copyright &y& Elsevier]
- Published
- 2002
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15. A theoretical multiscale treatment of protein–protein electron transfer: The ferredoxin/ferredoxin-NADP+ reductase and flavodoxin/ferredoxin-NADP+ reductase systems.
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Saen-oon, Suwipa, Cabeza de Vaca, Israel, Masone, Diego, Medina, Milagros, and Guallar, Victor
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CHARGE exchange , *PROTEINS , *FERREDOXINS , *PHOTOSYSTEMS , *FLAVODOXIN - Abstract
In the photosynthetic electron transfer (ET) chain, two electrons transfer from photosystem I to the flavin-dependent ferredoxin-NADP + reductase (FNR) via two sequential independent ferredoxin (Fd) electron carriers. In some algae and cyanobacteria (as Anabaena ), under low iron conditions, flavodoxin (Fld) replaces Fd as single electron carrier. Extensive mutational studies have characterized the protein–protein interaction in FNR/Fd and FNR/Fld complexes. Interestingly, even though Fd and Fld share the interaction site on FNR, individual residues on FNR do not participate to the same extent in the interaction with each of the protein partners, pointing to different electron transfer mechanisms. Despite of extensive mutational studies, only FNR/Fd X-ray structures from Anabaena and maize have been solved; structural data for FNR/Fld remains elusive. Here, we present a multiscale modelling approach including coarse-grained and all-atom protein–protein docking, the QM/MM e-Pathway analysis and electronic coupling calculations, allowing for a molecular and electronic comprehensive analysis of the ET process in both complexes. Our results, consistent with experimental mutational data, reveal the ET in FNR/Fd proceeding through a bridge-mediated mechanism in a dominant protein–protein complex, where transfer of the electron is facilitated by Fd loop-residues 40–49. In FNR/Fld, however, we observe a direct transfer between redox cofactors and less complex specificity than in Fd; more than one orientation in the encounter complex can be efficient in ET. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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16. Dynamics of the active site architecture in plant-type ferredoxin-NADP+ reductases catalytic complexes.
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Sánchez-Azqueta, Ana, Catalano-Dupuy, Daniela L., López-Rivero, Arleth, Tondo, María Laura, Orellano, Elena G., Ceccarelli, Eduardo A., and Medina, Milagros
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BINDING sites , *FERREDOXIN-NADP reductase , *ENZYME kinetics , *CATALYTIC activity , *HYDRIDE transfer reactions , *ANABAENA - Abstract
Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP + reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP + coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor–acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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17. External loops at the ferredoxin-NADP+ reductase protein–partner binding cavity contribute to substrates allocation.
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Sánchez-Azqueta, Ana, Martínez-Júlvez, Marta, Hervás, Manuel, Navarro, José A., and Medina, Milagros
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FERREDOXIN-NADP reductase , *PROTEIN binding , *BIOCHEMICAL substrates , *OXIDATION-reduction reaction , *CATALYSIS , *FLAVINS , *PYROPHOSPHATES - Abstract
Abstract: Ferredoxin-NADP+ reductase (FNR) is the structural prototype of a family of FAD-containing reductases that catalyze electron transfer between low potential proteins and NAD(P)+/H, and that display a two-domain arrangement with an open cavity at their interface. The inner part of this cavity accommodates the reacting atoms during catalysis. Loops at its edge are highly conserved among plastidic FNRs, suggesting that they might contribute to both flavin stabilization and competent disposition of substrates. Here we pay attention to two of these loops in Anabaena FNR. The first is a sheet–loop–sheet motif, loop102–114, that allocates the FAD adenosine. It was thought to determine the extended FAD conformation, and, indirectly, to modulate isoalloxazine electronic properties, partners binding, catalytic efficiency and even coenzyme specificity. The second, loop261–269, contains key residues for the allocation of partners and coenzyme, including two glutamates, Glu267 and Glu268, proposed as candidates to facilitate the key displacement of the C-terminal tyrosine (Tyr303) from its stacking against the isoalloxazine ring during the catalytic cycle. Our data indicate that the main function of loop102–114 is to provide the inter-domain cavity with flexibility to accommodate protein partners and to guide the coenzyme to the catalytic site, while the extended conformation of FAD must be induced by other protein determinants. Glu267 and Glu268 appear to assist the conformational changes that occur in the loop261–269 during productive coenzyme binding, but their contribution to Tyr303 displacement is minor than expected. Additionally, loop261–269 appears a determinant to ensure reversibility in photosynthetic FNRs. [Copyright &y& Elsevier]
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- 2014
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18. A hydrogen bond network in the active site of Anabaena ferredoxin-NADP+ reductase modulates its catalytic efficiency.
- Author
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Sánchez-Azqueta, Ana, Herguedas, Beatriz, Hurtado-Guerrero, Ramón, Hervás, Manuel, Navarro, José A., Martínez-Júlvez, Marta, and Medina, Milagros
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HYDROGEN bonding , *ANABAENA , *CATALYSIS , *FERREDOXIN-NADP reductase , *PHOTOSYNTHESIS , *ALLOXAZINE - Abstract
Abstract: Ferredoxin-nicotinamide–adenine dinucleotide phosphate (NADP+) reductase (FNR) catalyses the production of reduced nicotinamide–adenine dinucleotide phosphate (NADPH) in photosynthetic organisms, where its flavin adenine dinucleotide (FAD) cofactor takes two electrons from two reduced ferredoxin (Fd) molecules in two sequential steps, and transfers them to NADP+ in a single hydride transfer (HT) step. Despite the good knowledge of this catalytic machinery, additional roles can still be envisaged for already reported key residues, and new features are added to residues not previously identified as having a particular role in the mechanism. Here, we analyse for the first time the role of Ser59 in Anabaena FNR, a residue suggested by recent theoretical simulations as putatively involved in competent binding of the coenzyme in the active site by cooperating with Ser80. We show that Ser59 indirectly modulates the geometry of the active site, the interaction with substrates and the electronic properties of the isoalloxazine ring, and in consequence the electron transfer (ET) and HT processes. Additionally, we revise the role of Tyr79 and Ser80, previously investigated in homologous enzymes from plants. Our results probe that the active site of FNR is tuned by a H-bond network that involves the side-chains of these residues and that results to critical optimal substrate binding, exchange of electrons and, particularly, competent disposition of the C4n (hydride acceptor/donor) of the nicotinamide moiety of the coenzyme during the reversible HT event. [Copyright &y& Elsevier]
- Published
- 2014
- Full Text
- View/download PDF
19. The C-terminal extension of bacterial flavodoxin-reductases: Involvement in the hydride transfer mechanism from the coenzyme.
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Bortolotti, Ana, Sánchez-Azqueta, Ana, Maya, Celia M., Velázquez-Campoy, Adrián, Hermoso, Juan A., Medina, Milagros, and Cortez, Néstor
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C-terminal residues , *FLAVODOXIN , *REDUCTASES , *HYDRIDE transfer reactions , *COENZYMES , *RHODOBACTER capsulatus , *FLAVOPROTEINS , *LIGHT absorbance , *FLUORESCENCE spectroscopy - Abstract
Abstract: To study the role of the mobile C-terminal extension present in bacterial class of plant type NADP(H):ferredoxin reductases during catalysis, we generated a series of mutants of the Rhodobacter capsulatus enzyme (RcFPR). Deletion of the six C-terminal amino acids beyond alanine 266 was combined with the replacement A266Y, emulating the structure present in plastidic versions of this flavoenzyme. Analysis of absorbance and fluorescence spectra suggests that deletion does not modify the general geometry of FAD itself, but increases exposure of the flavin to the solvent, prevents a productive geometry of FAD:NADP(H) complex and decreases the protein thermal stability. Although the replacement A266Y partially coats the isoalloxazine from solvent and slightly restores protein stability, this single change does not allow formation of active charge-transfer complexes commonly present in the wild-type FPR, probably due to restraints of C-terminus pliability. A proton exchange process is deduced from ITC measurements during coenzyme binding. All studied RcFPR variants display higher affinity for NADP+ than wild-type, evidencing the contribution of the C-terminus in tempering a non-productive strong (rigid) interaction with the coenzyme. The decreased catalytic rate parameters confirm that the hydride transfer from NADPH to the flavin ring is considerably hampered in the mutants. Although the involvement of the C-terminal extension from bacterial FPRs in stabilizing overall folding and bent-FAD geometry has been stated, the most relevant contributions to catalysis are modulation of coenzyme entrance and affinity, promotion of the optimal geometry of an active complex and supply of a proton acceptor acting during coenzyme binding. [Copyright &y& Elsevier]
- Published
- 2014
- Full Text
- View/download PDF
20. Structural backgrounds for the formation of a catalytically competent complex with NADP(H) during hydride transfer in ferredoxin–NADP+ reductases
- Author
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Sánchez-Azqueta, Ana, Musumeci, Matías A., Martínez-Júlvez, Marta, Ceccarelli, Eduardo A., and Medina, Milagros
- Subjects
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NICOTINAMIDE adenine dinucleotide phosphate , *NADPH oxidase , *HYDRIDE transfer reactions , *FERREDOXIN-NADP reductase , *PEAS , *REDUCTION potential - Abstract
Abstract: The role of the highly conserved C266 and L268 of pea ferredoxin–NADP+ reductase (FNR) in formation of the catalytically competent complex of the enzyme with NADP(H) was investigated. Previous studies suggest that the volume of these side-chains, situated facing the side of the C-terminal Y308 catalytic residue not stacking the flavin isoalloxazine ring, may be directly involved in the fine-tuning of the catalytic efficiency of the enzyme. Wild-type pea FNR as well as single and double mutants of C266 and L268 residues were analysed by fast transient-kinetic techniques and their midpoint reduction potentials were determined. For the C266A, C266M and C266A/L268A mutants a significant reduction in the overall hydride transfer (HT) rates was observed along with the absence of charge-transfer complex formation. The HT rate constants for NADPH oxidation were lower than those for NADP+ reduction, reaching a 30-fold decrease in the double mutant. In agreement, these variants exhibited more negative midpoint potentials with respect to the wild-type enzyme. The three-dimensional structures of C266M and L268V variants were solved. The C266M mutant shows a displacement of E306 away from the relevant residue S90 to accommodate the bulky methionine introduced. The overall findings indicate that in FNR the volume of the residue at position 266 is essential to attain the catalytic architecture between the nicotinamide and isoalloxazine rings at the active site and, therefore, for an efficient HT process. In addition, flexibility of the 268–270 loop appears to be critical for FNR to achieve catalytically competent complexes with NADP(H). [Copyright &y& Elsevier]
- Published
- 2012
- Full Text
- View/download PDF
21. Modulating O2 Reactivity in a Fungal Flavoenzyme: INVOLVEMENT OF ARYL-ALCOHOL OXIDASE PHE-501 CONTIGUOUS TO CATALYTIC HISTIDINE.
- Author
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Hernández-Ortega, Aitor, Lucas, Fátima, Ferreira, Patricia, Medina, Milagros, Guallar, Victor, and Martínez, Angel T.
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FLAVOPROTEINS , *ALCOHOL oxidase , *HISTIDINE , *LIGNINS , *MUTAGENESIS - Abstract
Aryl-alcohol oxidase (AAO) is a flavoenzyme responsible for activation of O2 to H2O2 in fungal degradation of lignin. The AAO crystal structure shows a buried active site connected to the solvent by a hydrophobic funnel-shaped channel, with Phe-501 and two other aromatic residues forming a narrow bottleneck that prevents the direct access of alcohol substrates. However, ligand diffusion simulations show O2 access to the active site following this channel. Site-directed mutagenesis of Phe-501 yielded a F501A variant with strongly reduced O2 reactivity. However, a variant with increased reactivity, as shown by kinetic constants and steady-state oxidation degree, was obtained by substitution of Phe-501 with tryptophan. The high oxygen catalytic efficiency of F501W, ~2-fold that of native AAO and ~120-fold that of F501A, seems related to a higher O2 availability because the turnover number was slightly decreased with respect to the native enzyme. Free diffusion simulations of O2 inside the active-site cavity of AAO (and several in silico Phe-501 variants) yielded >60% O2 population at 3-4 Å from flavin C4a in F501W compared with 44% in AAO and only 14% in F501A. Paradoxically, the O2 reactivity of AAO decreased when the access channel was enlarged and increased when it was constricted by introducing a tryptophan residue. This is because the side chain of Phe-501, contiguous to the catalytic histidine (His-502 in AAO), helps to position O2 at an adequate distance from flavin C4a (and His-502 Nϵ). Phe-501 substitution with a bulkier tryptophan residue resulted in an increase in the O2 reactivity of this flavoenzyme. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
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22. Role of specific residues in coenzyme binding, charge–transfer complex formation, and catalysis in Anabaena ferredoxin NADP+-reductase
- Author
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Peregrina, José Ramón, Sánchez-Azqueta, Ana, Herguedas, Beatriz, Martínez-Júlvez, Marta, and Medina, Milagros
- Subjects
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ELECTRON donor-acceptor complexes , *FERREDOXIN-NADP reductase , *SITE-specific mutagenesis , *CHARGE exchange , *NICOTINAMIDE , *HYDRIDES , *CHEMICAL kinetics - Abstract
Abstract: Two transient charge–transfer complexes (CTC) form prior and upon hydride transfer (HT) in the reversible reaction of the FAD-dependent ferredoxin-NADP+ reductase (FNR) with NADP+/H, FNRox-NADPH (CTC-1), and FNRrd-NADP+ (CTC-2). Spectral properties of both CTCs, as well as the corresponding interconversion HT rates, are here reported for several Anabaena FNR site-directed mutants. The need for an adequate initial interaction between the 2′P-AMP portion of NADP+/H and FNR that provides subsequent conformational changes leading to CTC formation is further confirmed. Stronger interactions between the isoalloxazine and nicotinamide rings might relate with faster HT processes, but exceptions are found upon distortion of the active centre. Thus, within the analyzed FNR variants, there is no strict correlation between the stability of the transient CTCs formation and the rate of the subsequent HT. Kinetic isotope effects suggest that, while in the WT, vibrational enhanced modulation of the active site contributes to the tunnel probability of HT; complexes of some of the active site mutants with the coenzyme hardly allow the relative movement of isoalloxazine and nicotinamide rings along the HT reaction. The architecture of the WT FNR active site precisely contributes to reduce the stacking probability between the isoalloxazine and nicotinamide rings in the catalytically competent complex, modulating the angle and distance between the N5 of the FAD isoalloxazine and the C4 of the coenzyme nicotinamide to values that ensure efficient HT processes. [Copyright &y& Elsevier]
- Published
- 2010
- Full Text
- View/download PDF
23. Dual role of FMN in flavodoxin function: Electron transfer cofactor and modulation of the protein–protein interaction surface
- Author
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Frago, Susana, Lans, Isaias, Navarro, José A., Hervás, Manuel, Edmondson, Dale E., De la Rosa, Miguel A., Gómez-Moreno, Carlos, Mayhew, Stephen G., and Medina, Milagros
- Subjects
- *
CHARGE exchange , *PROTEIN-protein interactions , *PHOTOSYNTHESIS , *DIPOLE moments , *ANABAENA , *QUINONE , *VITAMIN B2 , *HYDROQUINONE - Abstract
Abstract: Flavodoxin (Fld) replaces Ferredoxin (Fd) as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP+ reductase (FNR). A number of Anabaena Fld (AnFld) variants with replacements at the interaction surface with FNR and PSI indicated that neither polar nor hydrophobic residues resulted critical for the interactions, particularly with FNR. This suggests that the solvent exposed benzenoid surface of the Fld FMN cofactor might contribute to it. FMN has been replaced with analogues in which its 7- and/or 8-methyl groups have been replaced by chlorine and/or hydrogen. The oxidised Fld variants accept electrons from reduced FNR more efficiently than Fld, as expected from their less negative midpoint potential. However, processes with PSI (including reduction of Fld semiquinone by PSI, described here for the first time) are impeded at the steps that involve complex re-arrangement and electron transfer (ET). The groups introduced, particularly chlorine, have an electron withdrawal effect on the pyrazine and pyrimidine rings of FMN. These changes are reflected in the magnitude and orientation of the molecular dipole moment of the variants, both factors appearing critical for the re-arrangement of the finely tuned PSI:Fld complex. Processes with FNR are also slightly modulated. Despite the displacements observed, the negative end of the dipole moment points towards the surface that contains the FMN, still allowing formation of complexes competent for efficient ET. This agrees with several alternative binding modes in the FNR:Fld interaction. In conclusion, the FMN in Fld not only contributes to the redox process, but also to attain the competent interaction of Fld with FNR and PSI. [Copyright &y& Elsevier]
- Published
- 2010
- Full Text
- View/download PDF
24. Aryl-alcohol Oxidase Involved in Lignin Degradation.
- Author
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Ferreira, Patricia, Hernandez-Ortega, Aitor, Herguedas, Beatriz, Martínez, Ángel T., and Medina, Milagros
- Subjects
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FUNGI , *OXIDATION-reduction reaction , *BIODEGRADATION , *PLEUROTUS , *LIGNINS , *HYDROGEN peroxide , *OXIDOREDUCTASES , *PHYSIOLOGY - Abstract
Aryl-alcohol oxidase (AAO) is a FAD-containing enzyme in the GMC (glucose-methanol-choline oxidase) family of oxidoreductases. AAO participates in fungal degradation of lignin, a process of high ecological and biotechnological relevance, by providing the hydrogen peroxide required by ligninolytic peroxidases. In the Pleurotus species, this peroxide is generated in the redox cycling of p-anisaldehyde, an extracellular fungal metabolite. In addition to p-anisyl alcohol, the enzyme also oxidizes other polyunsaturated primary alcohols. Its reaction mechanism was investigated here using p-anisyl alcohol and 2,4-hexadien-1-ol as two AAO model substrates. Steady state kinetic parameters and enzyme-monitored turnover were consistent with a sequential mechanism in which O2 reacts with reduced AAO before release of the aldehyde product. Pre-steady state analysis revealed that the AAO reductive half-reaction is essentially irreversible and rate limiting during catalysis. Substrate and solvent kinetic isotope effects under steady and presteady state conditions (the latter showing ∼9-fold slower enzyme reduction when α-bideuterated substrates were used, and ∼13-fold slower reduction when both substrate and solvent effects were simultaneously evaluated) revealed a synchronous mechanism in which hydride transfer from substrate a-carbon to FAD and proton abstraction from hydroxyl occur simultaneously. This significantly differs from the general mechanism proposed for other members of the GMC oxidoreductase family that implies hydride transfer from a previously stabilized substrate alkoxide. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
25. Flavodoxin: A compromise between efficiency and versatility in the electron transfer from Photosystem I to Ferredoxin-NADP+ reductase
- Author
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Goñi, Guillermina, Herguedas, Beatriz, Hervás, Manuel, Peregrina, José R., De la Rosa, Miguel A., Gómez-Moreno, Carlos, Navarro, José A., Hermoso, Juan A., Martínez-Júlvez, Marta, and Medina, Milagros
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BACTERIAL proteins , *CHARGE exchange , *FERREDOXIN-NADP reductase , *PROTEIN-protein interactions , *BIOENERGETICS - Abstract
Abstract: Under iron-deficient conditions Flavodoxin (Fld) replaces Ferredoxin in Anabaena as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP+ reductase (FNR). Several residues modulate the Fld interaction with FNR and PSI, but no one appears as specifically critical for efficient electron transfer (ET). Fld shows a strong dipole moment, with its negative end directed towards the flavin ring. The role of this dipole moment in the processes of interaction and ET with positively charged surfaces exhibited by PSI and FNR has been analysed by introducing single and multiple charge reversal mutations on the Fld surface. Our data confirm that in this system interactions do not rely on a precise complementary surface of the reacting molecules. In fact, they indicate that the initial orientation driven by the alignment of dipole moment of the Fld molecule with that of the partner contributes to the formation of a bunch of alternative binding modes competent for the efficient ET reaction. Additionally, the fact that Fld uses different interaction surfaces to dock to PSI and to FNR is confirmed. [Copyright &y& Elsevier]
- Published
- 2009
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26. Involvement of the Pyrophosphate and the 2'-Phosphate Binding Regions of Ferredoxin-NADP[sup +] Reductase in Coenzyme Specificity.
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Tejero, Jesús, Martínez-Júlvez, Marta, Mayoral, Tomás, Luquita, Alejandra, Sanz-Aparicio, Julia, Hermoso, Juan A., Hurley, John K., Tollin, Gordon, Gómez-Moreno, Carlos, and Medina, Milagros
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COENZYMES , *PYROPHOSPHATES , *PROTEIN binding , *GENETIC mutation - Abstract
Previous studies indicated that the determinants of coenzyme specificity in ferredoxin-NADP[sup +] reductase (FNR) from Anabaena are situated in the 2'-phosphate (2'-P) NADP[sup +] binding region, and also suggested that other regions must undergo structural rearrangements of the protein backbone during coenzyme binding. Among the residues involved in such specificity could be those located in regions where interaction with the pyrophosphate group of the coenzyme takes place, namely loops 155-160 and 261-268 in Anabaena FNR. In order to learn more about the coenzyme specificity determinants, and to better define the structural basis of coenzyme binding, mutations in the pyrophosphate and 2'-P binding regions of FNR have been introduced. Modification of the pyrophosphate binding region, involving residues Thr-155, Ala-160, and Leu-263, indicates that this region is involved in determining coenzyme specificity and that selected alterations of these positions produce FNR enzymes that are able to bind NAD[sup +]. Thus, our results suggest that slightly different structural rearrangements of the backbone chain in the pyrophosphate binding region might determine FNR specificity for the coenzyme. Combined mutations at the 2'-P binding region, involving residues Ser-223, Arg-224, Arg-233, and Tyr-235, in combination with the residues mentioned above in the pyrophosphate binding region have also been carried out in an attempt to increase the FNR affinity for NAD[sup +]/H. However, in most cases the analyzed mutants lost the ability for NADP[sup +]/H binding and electron transfer, and no major improvements were observed with regard to the efficiency of the reactions with NAD[sup +]/H. Therefore, our results confirm that determinants for coenzyme specificity in FNR are also situated in the pyrophosphate binding region and not only in the 2'-P binding region. Such observations also suggest that other regions of the protein, yet to be identified, might also be involved in this process. [ABSTRACT FROM AUTHOR]
- Published
- 2003
- Full Text
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27. Structure–function relationships in Anabaena ferredoxin/ferredoxin:NADP+ reductase electron transfer: insights from site-directed mutagenesis, transient absorption spectroscopy and X-ray crystallography
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Hurley, John K., Morales, Renaud, Martınez-Júlvez, Marta, Brodie, Tammy B., Medina, Milagros, Gómez-Moreno, Carlos, and Tollin, Gordon
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ANABAENA , *PHOTOSYNTHESIS , *BIOELECTRONICS - Abstract
The interaction between reduced Anabaena ferredoxin and oxidized ferredoxin:NADP+ reductase (FNR), which occurs during photosynthetic electron transfer (ET), has been investigated extensively in the authors'' laboratories using transient and steady-state kinetic measurements and X-ray crystallography. The effect of a large number of site-specific mutations in both proteins has been assessed. Many of the mutations had little or no effect on ET kinetics. However, non-conservative mutations at three highly conserved surface sites in ferredoxin (F65, E94 and S47) caused ET rate constants to decrease by four orders of magnitude, and non-conservative mutations at three highly conserved surface sites in FNR (L76, K75 and E301) caused ET rate constants to decrease by factors of 25–150. These residues were deemed to be critical for ET. Similar mutations at several other conserved sites in the two proteins (D67 in Fd; E139, L78, K72, and R16 in FNR) caused smaller but still appreciable effects on ET rate constants. A strong correlation exists between these results and the X-ray crystal structure of an Anabaena ferredoxin/FNR complex. Thus, mutations at sites that are within the protein–protein interface or are directly involved in interprotein contacts generally show the largest kinetic effects. The implications of these results for the ET mechanism are discussed. [Copyright &y& Elsevier]
- Published
- 2002
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28. Fast and slow biomembrane solubilizing detergents: Insights into their mechanism of action.
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Lete, Marta G., Monasterio, Bingen G., Collado, M. Isabel, Medina, Milagros, Sot, Jesús, Alonso, Alicia, and Goñi, Félix M.
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BIOCHEMICAL mechanism of action , *DETERGENTS , *MICELLAR solutions , *TRITON X-100 , *SODIUM sulfate , *CELL membranes - Abstract
• Detergents are empirically used in the study of cell membranes. • This work intends to explore the mechanisms of membrane solubilization by detergents. • The behaviour of 21 commonly used, chemically heterogeneous surfactants, has been examined. • Membrane solubilization by detergents is directly related to the surfactant transbilayer (flipping) ability. Detergents are water-soluble amphiphiles. Above a critical concentration they self-organize in micelles and in the presence of phospholipids mixed micelles are formed. Much information is available on the structure of these self-assemblies and on the thermodynamics of their formation. The aim of this study was to deepen our understanding of the mechanisms of solubilization. Solubilization of lipid vesicles made of egg phosphatidylcholine (PC) by twenty one commercially available, structurally heterogeneous detergents, has been assessed by a decrease in turbidity of the vesicle suspension. Both steady-state and time-resolved measurements have been performed. The results show that the detergents under study fall into one of two categories, namely fast-solubilizing and slow-solubilizing detergents. This categorization is independent of detergent concentration, i.e. a "slow" cannot be converted into a "fast" surfactant by increasing its bulk concentration. 31P-NMR spectra indicate that slow-acting detergents cause either a gradual, monotonic micellization of bilayers (sodium dodecyl sulphate), or formation of more complex, perhaps non-lamellar, non-micellar intermediates (dodecylmaltoside). In contrast, fast detergents (e.g. Triton X-100) cause lysis and reassembly of vesicles before bulk solubilization takes place. These results support the idea that membrane solubilization by detergents is rapid only when surfactant transbilayer (flipping) motion is easy. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
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29. Towards the competent conformation for catalysis in the ferredoxin-NADP+ reductase from the Brucella ovis pathogen.
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Pérez-Amigot, Daniel, Taleb, Víctor, Boneta, Sergio, Anoz-Carbonell, Ernesto, Sebastián, María, Velázquez-Campoy, Adrián, Polo, Víctor, Martínez-Júlvez, Marta, and Medina, Milagros
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NICOTINAMIDE , *CATALYSIS , *ELECTRON donor-acceptor complexes , *MOLECULAR dynamics , *NICOTINAMIDE adenine dinucleotide phosphate - Abstract
Brucella ovis encodes a bacterial subclass 1 ferredoxin-NADP(H) reductase (BoFPR) that, by similarity with other FPRs, is expected either to deliver electrons from NADPH to the redox-based metabolism and/or to oxidize NADPH to regulate the soxRS regulon that protects bacteria against oxidative damage. Such potential roles for the pathogen survival under infection conditions make of interest to understand and to act on the BoFPR mechanism. Here, we investigate the NADP+/H interaction and NADPH oxidation by hydride transfer (HT) to BoFPR. Crystal structures of BoFPR in free and in complex with NADP+ hardly differ. The latter shows binding of the NADP+ adenosine moiety, while its redox-reactive nicotinamide protrudes towards the solvent. Nonetheless, pre-steady-state kinetics show formation of a charge-transfer complex (CTC-1) prior to the hydride transfer, as well as conversion of CTC-1 into a second charge-transfer complex (CTC-2) concomitantly with the HT event. Thus, during catalysis nicotinamide and flavin reacting rings stack. Kinetic data also identify the HT itself as the rate limiting step in the reduction of BoFPR by NADPH, as well as product release limiting the overall reaction. Using all-atom molecular dynamics simulations with a thermal effect approach we are able to visualise a potential transient catalytically competent interaction of the reacting rings. Simulations indicate that the architecture of the FAD folded conformation in BoFPR might be key in catalysis, pointing to its adenine as an element to orient the reactive atoms in conformations competent for HT. Unlabelled Image • Subclass 1 BoFPR oxidizes NADPH through formation of two charge transfer complexes. • NADPH nicotinamide and FAD isoalloxazine rings stack during catalysis. • X-ray diffraction fails envisaging catalytic geometries or how they are attained. • MD allows predicting potential competent interactions of the reacting rings. • The adenine moiety of FAD might facilitate the overall catalytic event in BoFPR. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
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