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Milagros Medina - One of the best experts on this subject based on the ideXlab platform.
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Specific Features for the Competent Binding of Substrates at the FMN Adenylyltransferase Site of FAD Synthase from Corynebacterium ammoniagenes.
International Journal of Molecular Sciences, 2019Co-Authors: Sonia Arilla-luna, Ana Serrano, Milagros MedinaAbstract:Bifunctional FAD synthases (FADSs) catalyze FMN (flavin mononucleotide) and FAD (flavinadenine dinucleotide) biosynthesis at their C-riboflavin kinase (RFK) and N-FMN:adenylyltransferase (FMNAT) modules, respectively. Biophysical properties and requirements for their FMNAT activity differ among species. Here, we evaluate the relevance of the integrity of the binding site of the Isoalloxazine of flavinic substrates for FMNAT catalysis in Corynebacterium ammoniagenes FADS (CaFADS). We have substituted P56 and P58, belonging to a conserved motif, as well as L98. These residues shape the Isoalloxazine FMNAT site, although they are not expected to directly contact it. All substitutions override enzyme ability to transform substrates at the FMNAT site, although most variants are able to bind them. Spectroscopic properties and thermodynamic parameters for the binding of ligands indicate that mutations alter their interaction modes. Substitutions also modulate binding and kinetic properties at the RFK site, evidencing the crosstalk of different protomers within CaFADS assemblies during catalysis. In conclusion, despite the FMNAT site for the binding of substrates in CaFADS appearing as a wide open cavity, it is finely tuned to provide the competent binding conformation of substrates. In particular, P56, P58 and L98 shape the Isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis.
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understanding the fmn cofactor chemistry within the anabaena flavodoxin environment
Biochimica et Biophysica Acta, 2012Co-Authors: Isaias Lans, Susana Frago, Milagros MedinaAbstract: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.
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role of specific residues in coenzyme binding charge transfer complex formation and catalysis in anabaena ferredoxin nadp reductase
Biochimica et Biophysica Acta, 2010Co-Authors: Jose Ramon Peregrina, Ana Sanchezazqueta, Beatriz Herguedas, Marta Martinezjulvez, Milagros MedinaAbstract: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, FNR(ox)-NADPH (CTC-1), and FNR(rd)-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.
Genji Kurisu - One of the best experts on this subject based on the ideXlab platform.
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kinetic and structural insight into a role of the re face tyr328 residue of the homodimer type ferredoxin nadp oxidoreductase from rhodopseudomonas palustris in the reaction with nadp nadph
Biochimica et Biophysica Acta, 2020Co-Authors: Genji Kurisu, Norifumi MurakiAbstract:Abstract Among the thioredoxin reductase-type ferredoxin-NAD(P)+ oxidoreductase (FNR) family, FNR from photosynthetic purple non‑sulfur bacterium Rhodopseudomonas palustris (RpFNR) is distinctive because the predicted residue on the re-face of the Isoalloxazine ring portion of the FAD prosthetic group is a tyrosine. Here, we report the crystal structure of wild type RpFNR and kinetic analyses of the reaction of wild type, and Y328F, Y328H and Y328S mutants with NADP+/NADPH using steady state and pre-steady state kinetic approaches. The obtained crystal structure of wild type RpFNR confirmed the presence of Tyr328 on the re-face of the Isoalloxazine ring of the FAD prosthetic group through the unique hydrogen bonding of its hydroxyl group. In the steady state assays, the substitution results in the decrease of Kd for NADP+ and KM for NADPH in the diaphorase assay; however, the kcat values also decreased significantly. In the stopped-flow spectrophotometry, mixing oxidized RpFNRs with NADPH and reduced RpFNRs with NADP+ resulted in rapid charge transfer complex formation followed by hydride transfer. The observed rate constants for the hydride transfer in both directions were comparable (>400 s−1). The substitution did not drastically affect the rate of hydride transfer, but substantially slowed down the subsequent release and re-association of NADP+/NADPH in both directions. The obtained results suggest that Tyr328 stabilizes the stacking of C-terminal residues on the Isoalloxazine ring portion of the FAD prosthetic group, which impedes the access of NADP+/NADPH on the Isoalloxazine ring portions, in turn, enhancing the release of the NADP+/NADPH and/or reaction with electron transfer proteins.
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crystal structure of ferredoxin nad p reductase from the green sulfur bacterium chlorobaculum tepidum
2013Co-Authors: Daisuke Seo, Norifumi Muraki, Tomoo Shiba, Takeshi Sakurai, Genji KurisuAbstract:Green sulfur bacterium Chlorobaculum tepidum contains a novel type of ferredoxin-NAD(P)+ reductase (FNR) with high amino acid sequence homology to the NADPH-thioredoxin reductase (TdR) from prokaryotes. In this study, we determine the crystal structure of C. tepidum FNR by X-ray crystallography. C. tepidum FNR retains its structural topology with E. coli TdR but possesses several characteristic features that is absent in TdR. Each protomer is composed of two nucleotide binding domains, FAD-binding and NAD(P)+-binding. The two domains are connected by a hinge region. Homo-dimeric C. tepidum FNR shows an asymmetric domain orientation between two protomers. The observed C-terminal sub-domain covers the re-face of the Isoalloxazine ring of FAD prosthetic group. The C-terminal sub-domain includes the stacking Phe337 on the reface of the Isoalloxazine ring of the FAD. On the si-face, Tyr57 residue is stacked on. The two stacking ring systems are positioned almost parallel with respect to Isoalloxazine ring at a distance of 3.5 A. Such a configuration of stacking of two aromatic rings is absent in TdR but found in plastid-type FNRs, suggesting these structural characteristics are indispensable for the FNR reaction. To elucidate the function of these structural characteristics, mutational analysis was performed.
Zhen Shen - One of the best experts on this subject based on the ideXlab platform.
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towards modelling light processes of blue light photoreceptors pyrene Isoalloxazine flavin phenothiazine triad electrochemical photophysical investigations and quantum chemical calculations
Physical Chemistry Chemical Physics, 2003Co-Authors: Zhen Shen, Roman Prochazka, Jorg Daub, Norbert Fritz, Nursel Acar, Siegfried SchneiderAbstract:The triad 6 containing the phenothiazine–Isoalloxazine couple as donor–acceptor redox unit and pyrene as antenna absorbing in the UV-A region has been designed to mimic the light processes of natural photoreceptors. By cyclic voltammetry it is shown that the redox chemistry of the three subunits of triad 6 behave almost independently, indicating no electronic coupling between the subunits in the ground state. Triad 6 exhibits three accessible redox states with one oxidation and two reduction waves due to the formation of the phenothiazine radical cation and Isoalloxazine and pyrene radical anions. UV/Vis/NIR spectroelectrochemistry reveals the generation of the protonated Isoalloxazine dianion on reduction which is formed in the non-polar solvent in a reduction–protonation–reduction step (two-electron transfer process) and which is attributed to intermolecular proton transfer from the amide group to the electrochemically reduced Isoalloxazine radical anion. Evidences for the photoinduced energy and electron transfer within the triad are provided by steady state and time-resolved absorption and fluorescence measurements. Spectroscopic studies displayed that upon excitation the pyrene emission was dramatically quenched in the dyad 4. This is most likely due to the energy transfer from pyrene to the Isoalloxazine units as the absorption band of Isoalloxazine overlaps with the pyrene emission band leading most likely to a CT state of the Isoalloxazine/phenothiazine type. Quenching of the phenothiazine fluorescence in triad 6 was also ascribed to the spectroscopic overlap between the emission spectrum of phenothiazine and absorption spectrum of Isoalloxazine. Again, photoinduced electron transfer from phenothiazine to Isoalloxazine is expected to be the cause for the quenching of the Isoalloxazine emission in the dyad 5. Molecular orbital calculations for compound 5 showed a complete electron transfer from phenothiazin to Isoalloxazine.
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mimicking dye based functions of natural blue light photoreceptors by studying photoinduced energy and electron transfer in a pyrene Isoalloxazine flavin phenothiazine triad
Chemical Communications, 2002Co-Authors: Zhen Shen, Joerg Strauss, Joerg DaubAbstract:An artificial system containing phenothiazine as electron donor, Isoalloxazine as flavinoid redox-mediator and pyrene as antenna has been built up in order to model photoinduced energy and electron transfer processes of natural blue-light photoreceptors.
Richard P Swenson - One of the best experts on this subject based on the ideXlab platform.
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mechanism of flavin mononucleotide cofactor binding to the desulfovibrio vulgaris flavodoxin 1 kinetic evidence for cooperative effects associated with the binding of inorganic phosphate and the 5 phosphate moiety of the cofactor
Biochemistry, 2003Co-Authors: Tracey Arnold Murray, Richard P SwensonAbstract:The pathway(s) by which the flavin cofactor binds to the apoflavoprotein is the subject of some debate. The crystal and NMR structures of several different flavodoxins have provided some insight, although there is disagreement about the location of the initial interaction between the flavin mononucleotide (FMN) and the apoflavodoxin and the degree of protein conformational change associated with cofactor binding [Genzor, C. G., Perales-Alcon, A., Sancho, J., and Romero, A. (1996) Nat. Struct. Biol. 3, 329-332; Steensma, E., and van Mierlo, C. P. M. (1998) J. Mol. Biol. 282, 653-666]. Binding kinetics using stopped-flow spectrofluorimetry and phosphate competition studies were used to develop a model for flavin binding to the flavodoxin from Desulfovibrio vulgaris. In the presence of phosphate, the time course of fluorescence quenching associated with FMN binding to apoflavodoxin was biphasic, whereas riboflavin, which lacks the 5'-phosphate group of FMN, displayed monophasic binding kinetics. When the concentration of phosphate in solution was increased, the FMN binding rates of the two phases behaved differently; the rate of one phase decreased, while the rate of the other increased. A similar increase in the single phase associated with riboflavin binding was also observed. This has led to the following model. The binding of the flavin Isoalloxazine ring to its subsite is dependent on the presence of a phosphate group in the phosphate-binding subsite. When phosphate is in the buffer solution, FMN can bind in either of two ways: by the initial insertion of the 5'-phosphate group followed by ring binding or, when inorganic phosphate from solution is bound, the insertion of the Isoalloxazine ring first. Riboflavin, which lacks the phosphate moiety of FMN, binds only in the presence of inorganic phosphate, presumably due to the binding of this group in the phosphate-binding subsite. These results suggest that cooperative interactions exist between the phosphate subsite and the ring-binding region in the D. vulgaris flavodoxin that are necessary for Isoalloxazine ring binding.
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mechanism of flavin mononucleotide cofactor binding to the desulfovibrio vulgaris flavodoxin 2 evidence for cooperative conformational changes involving tryptophan 60 in the interaction between the phosphate and ring binding subsites
Biochemistry, 2003Co-Authors: Tracey Arnold Murray, Mark P Foster, Richard P SwensonAbstract:A mechanism has been proposed for the binding of flavin mononucleotide (FMN) and riboflavin to the apoflavodoxin from Desulfovibrio vulgaris [Murray, T. A., and Swenson, R. P. (2003) Biochemistry 42, 2307-2316]. In this model, the binding of the flavin Isoalloxazine ring is dependent on the presence of a phosphate moiety in the phosphate-binding subsite, suggesting a cooperative interaction between that region and the ring-binding subsite. In the absence of inorganic phosphate, FMN can bind through the initial association of its 5'-phosphate group in the phosphate-binding subsite followed by insertion of the flavin ring. Because riboflavin lacks the 5'-phosphate group, it is unable to bind to this apoprotein in the absence of inorganic phosphate in solution. However, inorganic phosphate can enhance the rate of ring binding by occupying the phosphate-binding subsite. In this paper, NMR, near-UV circular dichroism (CD), and fluorescence spectroscopy provide evidence for a phosphate-induced conformational change within the Isoalloxazine ring-binding subsite. Phosphate-dependent changes in the chemical shifts of 22 amide groups were observed in (1)H-(15)N HSQC NMR spectra. The majority of these groups are proximal to the phosphate-binding subsite or the loops that constitute the Isoalloxazine ring-binding site. Also, a phosphate-dependent change in the environment or position of the Trp60 side chain was apparent in the NMR data and was confirmed by associated changes in the near-UV CD and tryptophan fluorescence spectra when compared to the spectra of the W60A mutant. These data suggest that phosphate, either the 5'-phosphate of the FMN or inorganic phosphate from solution, facilitates the movement of the side chain of Trp60 out of the Isoalloxazine ring-binding site and other associated conformational changes, creating a population of apoflavodoxin that is capable of binding the Isoalloxazine ring. This conformational switch may explain why some apoflavodoxins cannot bind riboflavin and also supports the "aromatic gate" model proposed from the crystal structure of the Anabaena apoflavodoxin [Genzor, C. G., Perales-Alcon, A., Sancho, J., and Romero, A. (1996) Nat. Struct. Biol. 3, 329-332].
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site directed mutagenesis of tyrosine 98 in the flavodoxin from desulfovibrio vulgaris hildenborough regulation of oxidation reduction properties of the bound fmn cofactor by aromatic solvent and electrostatic interactions
Biochemistry, 1994Co-Authors: Richard P Swenson, Grigorios KreyAbstract:The contributions made by tyrosine-98 in establishing the redox properties of the flavodoxin from Desulfovibrio vulgaris were investigated by substituting a number of amino acids at this position using site-directed mutagenesis. Tyr98, which makes extensive van der Waals contacts with the Isoalloxazine ring of the flavin mononucleotide cofactor, is often found in the cofactor binding site of flavodoxins and related flavoproteins. Solution studies suggest that tyrosine may assist in the stabilization of the neutral flavin semiquinone through preferential complex formation relative to the other oxidation states. In this study, the midpoint potentials of the oxidized/semiquinone couple of the Y98W and Y98F mutants were found to be very similar to the wild-type flavodoxin. However, significantly more negative midpoint potentials (by 25-60 mV) were observed in the Y98A, Y98H, and Y98R mutants. These results imply that it is the general apolar environment provided by the aromatic amino acids rather than preferential affinities suggested by solution studies that is at least partially responsible for the thermodynamic stabilization of the neutral flavin semiquinone in this flavodoxin. The midpoint potential of the semiquinone/hydroquinone couple is profoundly dependent on the properties of the amino acid at this position. Compared to phenylalanine, the more electron-rich aromatic side chains of tryptophan and tyrosine decrease the midpoint potential of this couple by 30-40 mV. Greater solvent exposure of the Isoalloxazine ring in the Y98A mutant increases the midpoint potential by 140 mV relative to wild type. The positively charged amino acids increase the midpoint potential of this couple by > 180 mV, most probably through favorable electrostatic interactions with the flavin hydroquinone anion. These observations strongly support the proposition that the functional role of the electron-rich, apolar aromatic amino acid residues adjacent to the flavin Isoalloxazine ring is to substantially destabilize the flavin hydroquinone anion, resulting in the very low oxidation-reduction potentials for the semiquinone/hydroquinone couple that typify the flavodoxin family.
Norifumi Muraki - One of the best experts on this subject based on the ideXlab platform.
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kinetic and structural insight into a role of the re face tyr328 residue of the homodimer type ferredoxin nadp oxidoreductase from rhodopseudomonas palustris in the reaction with nadp nadph
Biochimica et Biophysica Acta, 2020Co-Authors: Genji Kurisu, Norifumi MurakiAbstract:Abstract Among the thioredoxin reductase-type ferredoxin-NAD(P)+ oxidoreductase (FNR) family, FNR from photosynthetic purple non‑sulfur bacterium Rhodopseudomonas palustris (RpFNR) is distinctive because the predicted residue on the re-face of the Isoalloxazine ring portion of the FAD prosthetic group is a tyrosine. Here, we report the crystal structure of wild type RpFNR and kinetic analyses of the reaction of wild type, and Y328F, Y328H and Y328S mutants with NADP+/NADPH using steady state and pre-steady state kinetic approaches. The obtained crystal structure of wild type RpFNR confirmed the presence of Tyr328 on the re-face of the Isoalloxazine ring of the FAD prosthetic group through the unique hydrogen bonding of its hydroxyl group. In the steady state assays, the substitution results in the decrease of Kd for NADP+ and KM for NADPH in the diaphorase assay; however, the kcat values also decreased significantly. In the stopped-flow spectrophotometry, mixing oxidized RpFNRs with NADPH and reduced RpFNRs with NADP+ resulted in rapid charge transfer complex formation followed by hydride transfer. The observed rate constants for the hydride transfer in both directions were comparable (>400 s−1). The substitution did not drastically affect the rate of hydride transfer, but substantially slowed down the subsequent release and re-association of NADP+/NADPH in both directions. The obtained results suggest that Tyr328 stabilizes the stacking of C-terminal residues on the Isoalloxazine ring portion of the FAD prosthetic group, which impedes the access of NADP+/NADPH on the Isoalloxazine ring portions, in turn, enhancing the release of the NADP+/NADPH and/or reaction with electron transfer proteins.
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crystal structure of ferredoxin nad p reductase from the green sulfur bacterium chlorobaculum tepidum
2013Co-Authors: Daisuke Seo, Norifumi Muraki, Tomoo Shiba, Takeshi Sakurai, Genji KurisuAbstract:Green sulfur bacterium Chlorobaculum tepidum contains a novel type of ferredoxin-NAD(P)+ reductase (FNR) with high amino acid sequence homology to the NADPH-thioredoxin reductase (TdR) from prokaryotes. In this study, we determine the crystal structure of C. tepidum FNR by X-ray crystallography. C. tepidum FNR retains its structural topology with E. coli TdR but possesses several characteristic features that is absent in TdR. Each protomer is composed of two nucleotide binding domains, FAD-binding and NAD(P)+-binding. The two domains are connected by a hinge region. Homo-dimeric C. tepidum FNR shows an asymmetric domain orientation between two protomers. The observed C-terminal sub-domain covers the re-face of the Isoalloxazine ring of FAD prosthetic group. The C-terminal sub-domain includes the stacking Phe337 on the reface of the Isoalloxazine ring of the FAD. On the si-face, Tyr57 residue is stacked on. The two stacking ring systems are positioned almost parallel with respect to Isoalloxazine ring at a distance of 3.5 A. Such a configuration of stacking of two aromatic rings is absent in TdR but found in plastid-type FNRs, suggesting these structural characteristics are indispensable for the FNR reaction. To elucidate the function of these structural characteristics, mutational analysis was performed.
