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Hamid M Said - One of the best experts on this subject based on the ideXlab platform.
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riboflavin transport by rabbit renal brush border membrane vesicles
Biochimica et Biophysica Acta, 1997Co-Authors: Norimoto Yanagawa, Hamid M SaidAbstract:The present study examined riboflavin (RF) uptake by isolated rabbit renal brush border membrane (BBM). RF uptake was linear for up to 30 s and leveled off thereafter reaching an equilibrium with longer incubation. Studies on RF uptake as a function of incubation medium osmolarity indicated that the uptake was the results of transport (61.4%) into the intravesicular space as well as binding (38.6%) to membrane surfaces. The process of RF uptake was saturable as a function of substrate concentration with an apparent Km of 25.7 +/- 7.6 microM and Vmax of 75.6 +/- 14.7 pmol/mg protein/10 s. cis-Addition of unlabeled RF and its structural analogues, lumiflavin and lumichrome, inhibited the uptake of [3H]RF significantly, indicating the involvement of a carrier-mediated process in RF uptake by renal BBM. RF uptake by renal BBM was partly Na+-dependent so that when Na+ was replaced by potassium, choline, lithium or tetramethylammonium, the RF uptake was reduced to ca. 60% of the control. This Na+-dependency was unlikely to be due to Na+-cotransport mechanism because RF uptake occurred without the characteristic 'overshoot' phenomenon as for other Na+-cotransport systems and the elimination of transmembrane Na+-gradient by preloading Na+ to the intravesicular space did not affect RF uptake. In contrast, removal of Na+ eliminated the binding component of RF uptake, suggesting the requirement of Na+ for RF binding to BBM. The RF uptake was not affected when extravesicular pH was varied within the physiological pH range of 6.5 to 8.5. No effect on BBM [3H]RF uptake was found when the transmembrane electrical potential was altered by either the presence of anions with different membrane permeability (Cl- = NO3- > SO4- > gluconate-) or by using nigericin (10 microg/mg protein) with an outwardly or inwardly directed transmembrane K+ gradient. The uptake of RF by BBM vesicles was, however, inhibited by probenecid and organic anion transport inhibitors, 4,4-diiso-thiocyanatostilbene-2,2-disulfonic acid (DIDS, 1 mM) and 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS, 1 mM). In summary, these results demonstrate the existence of a membrane-associated, and organic anion inhibitor-sensitive, carrier system for RF uptake by renal BBM.
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mechanism of transport of riboflavin in rabbit intestinal brush border membrane vesicles
Experimental Biology and Medicine, 1993Co-Authors: Hamid M Said, Reza Mohammadkhani, Eric MccloudAbstract:Uptake of luminal riboflavin (RF) into the absorptive cells of rabbit small intestine was examined using purified brush border membrane vesicle (BBMV) preparations. These preparations were used in order to eliminate the interference of intracellular metabolism that occurs to the RF molecule during absorption. Uptake of RF by BBMV was found to be mainly (> 76%) the result of transport of the vitamin into the intracellular space with less binding to membrane surfaces. All 3H radioactivity that appeared in the intravesicular space after incubation with [3H]RF was found to be in the form of intact RF. Uptake of RF with time was independent of the presence or absence of a Na+ or a K+ gradient (out > in) and occurred without transaccumulation of the substrate in the intravesicular space. Furthermore, changing the incubation buffer pH showed minimal effect on RF uptake. When examined as a function of concentration, the initial rate of RF uptake was found to be saturable both in jejunal and ileal BBMV with an apparent Km of 7.24 +/- 1.06 and 8.88 +/- 0.90 microM and Vmax of 24.31 +/- 1.48 and 34.24 +/- 1.55 pmol/mg protein/5 sec, respectively. Unlabeled RF and the related compounds lumiflavin, 8-aminoriboflavin, isoriboflavin, and lumichrome all inhibited (but to different degrees) the uptake of physiologic concentration of [3H]RF. On the other hand, 8-hydroxyriboflavin, lumazine, and D-ribose all failed to inhibit [3H]RF uptake. Similarly, the membrane transport inhibitors DIDS, SITS, and furosemide all failed to inhibit [3H]RF uptake. The uptake of RF was found to be insensitive to changes in the transmembrane electrical potential, as shown by studies with anion substitution and valinomycin K(+)-induced negative or positive intravesicular potential methodologies. These results indicate that RF uptake by rabbit intestinal BBMV occurs via a carrier-mediated system that is Na+ independent in nature and transports the substrate by an electroneutral process. The role of this system in the overall absorption process of RF is discussed.
Otto Dopfer - One of the best experts on this subject based on the ideXlab platform.
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effect of alkali ions on optical properties of flavins vibronic spectra of cryogenic m lumiflavin complexes m li cs
Faraday Discussions, 2019Co-Authors: David Muller, Pablo Nieto, Mitsuhiko Miyazaki, Otto DopferAbstract:Flavin compounds are frequently used by nature in photochemical processes because of their unique optical properties which can be strongly modulated by the surrounding environment such as solvation or coordination with metal ions. Herein, we employ vibronic photodissociation spectroscopy of cryogenic M+LF complexes composed of lumiflavin (LF, C13H12N4O2), the parent molecule of the flavin family, and alkali ions (M = Li–Cs) to characterize the strong impact of metalation on the electronic properties of the LF chromophore. With the aid of time-dependent density functional theory calculations (PBE0/cc-pVDZ) coupled to multidimensional Franck–Condon simulations, the visible photodissociation (VISPD) spectra of M+LF ions recorded in the 500–570 nm range are assigned to the S1 ← S0 (ππ*) transitions into the first optically bright S1 state of the lowest-energy M+LF(O4+) isomers. In this O4+ structure, M+ binds in a bent chelate to the lone pairs of both the O4 and the N5 atom of LF. Charge reorganization induced by S1 excitation strongly enhances the interaction between M+ and LF at this binding site, leading to substantial red shifts in the S1 absorption of the order of 10–20% (e.g., from 465 nm in LF to 567 nm in Li+LF). This strong change in M+⋯LF interaction strength in M+LF(O4+) upon ππ* excitation can be rationalized by the orbitals involved in the S1 ← S0 transition and causes strong vibrational activity. In particular, progressions in the intermolecular bending and stretching modes provide an accurate measure of the strength of the M+⋯LF bond. In contrast to the experimentally identified O4+ ions, the predicted S1 origins of other low-energy M+LF isomers, O2+ and O2, are slightly blue-shifted from the S1 of LF, demonstrating that the electronic properties of metalated LF not only drastically change with the size of the metal ion but also with its binding site.
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irmpd spectroscopy of metalated flavins structure and bonding of lumiflavin complexes with alkali and coinage metal ions
Journal of Physical Chemistry A, 2016Co-Authors: Pablo Nieto, Alan Gunther, Giel Berden, Jos Oomens, Otto DopferAbstract:Flavins are a fundamental class of biomolecules, whose photochemical properties strongly depend on their environment and their redox and metalation state. Infrared multiphoton dissociation (IRMPD) spectra of mass-selected isolated metal–lumiflavin ionic complexes (M+LF) are analyzed in the fingerprint range (800–1830 cm–1) to determine the bonding of lumiflavin with alkali (M = Li, Na, K, Cs) and coinage (M = Cu, Ag) metal ions. The complexes are generated in an electrospray ionization source coupled to an ion cyclotron resonance mass spectrometer and the IR free electron laser FELIX. Vibrational and isomer assignments of the IRMPD spectra are accomplished by comparison to quantum chemical calculations at the B3LYP/cc-pVDZ level, yielding structure, binding energy, bonding mechanism, and spectral properties of the complexes. The most stable binding sites identified in the experiments involve metal bonding to the oxygen atoms of the two available CO groups of LF. Hence, CO stretching frequencies are a sens...
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IRMPD Spectroscopy of Metalated Flavins: Structure and Bonding of Lumiflavin Complexes with Alkali and Coinage Metal Ions
2016Co-Authors: Pablo Nieto, Jos Oomens, Giel Berden, Alan Günther, Otto DopferAbstract:Flavins are a fundamental class of biomolecules, whose photochemical properties strongly depend on their environment and their redox and metalation state. Infrared multiphoton dissociation (IRMPD) spectra of mass-selected isolated metal–lumiflavin ionic complexes (M+LF) are analyzed in the fingerprint range (800–1830 cm–1) to determine the bonding of lumiflavin with alkali (M = Li, Na, K, Cs) and coinage (M = Cu, Ag) metal ions. The complexes are generated in an electrospray ionization source coupled to an ion cyclotron resonance mass spectrometer and the IR free electron laser FELIX. Vibrational and isomer assignments of the IRMPD spectra are accomplished by comparison to quantum chemical calculations at the B3LYP/cc-pVDZ level, yielding structure, binding energy, bonding mechanism, and spectral properties of the complexes. The most stable binding sites identified in the experiments involve metal bonding to the oxygen atoms of the two available CO groups of LF. Hence, CO stretching frequencies are a sensitive indicator of both the metal binding site and the metal bond strength. More than one isomer is observed for M = Li, Na, and K, and the preferred CO binding site changes with the size of the alkali ion. For Cs+LF, only one isomer is identified, although the energies of the two most stable structures differ by less than 7 kJ/mol. While the M+–LF bonds for alkali ions are mainly based on electrostatic forces, substantial covalent contributions lead to stronger bonds for the coinage metal ions. Comparison between lumiflavin and lumichrome reveals substantial differences in the metal binding motifs and interactions due to the different flavin structures
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probing protonation sites of isolated flavins using ir spectroscopy from lumichrome to the cofactor flavin mononucleotide
ChemPhysChem, 2014Co-Authors: Judith Langer, Alan Gunther, Sophie Seidenbecher, Giel Berden, Jos Oomens, Otto DopferAbstract:Infrared spectra of the isolated protonated flavin molecules lumichrome, lumiflavin, riboflavin (vitamin B-2), and the biologically important cofactor flavin mononucleotide are measured in the fingerprint region (600-1850 cm(-1)) by means of IR multiple-photon dissociation (IRMPD) spectroscopy. Using density functional theory calculations, the geometries, relative energies, and linear IR absorption spectra of several low-energy isomers are calculated. Comparison of the calculated IR spectra with the measured IRMPD spectra reveals that the N10 substituent on the isoalloxazine ring influences the protonation site of the flavin. Lumichrome, with a hydrogen substituent, is only stable as the N1-protonated tautomer and protonates at N5 of the pyrazine ring. The presence of the ribityl unit in riboflavin leads to protonation at N1 of the pyrimidinedione moiety, and methyl substitution in lumiflavin stabilizes the tautomer that is protonated at O2. In contrast, flavin mononucleotide exists as both the O2- and N1-protonated tautomers. The frequencies and relative intensities of the two C=O stretch vibrations in protonated flavins serve as reliable indicators for their protonation site.
Robert J. Stanley - One of the best experts on this subject based on the ideXlab platform.
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charge redistribution in oxidized and semiquinone e coli dna photolyase upon photoexcitation stark spectroscopy reveals a rationale for the position of trp382
Journal of the American Chemical Society, 2009Co-Authors: Goutham Kodali, Salim U Siddiqui, Robert J. StanleyAbstract:The electronic structure of the two lowest excited electronic states of FAD and FADHin folate- depleted E. coli DNA photolyase (PLOX and PLSQ, respectively) was measured using absorption Stark spectroscopy. The experimental analysis was supported by TDDFT calculations of both the charge redistribution and the difference dipole moments for the transitions of both oxidation states using lumiflavin as a model. The difference dipole moments and polarizabilities for PLOX are similar to those obtained in our previous work for flavins in simple solvents and in an FMN-containing flavoprotein. No such comparison can be made for PLSQ, as we believe this to be the first experimental report of the direction and magnitude of excited-state charge redistribution in any flavosemiquinone. The picture that emerges from these studies is discussed in the context of electron transfer in photolyase, particularly for the semiquinone photoreduction process, which involves nearby tryptophan residues as electron donors. The direction of charge displacement derived from an analysis of the Stark spectra rationalizes the positioning of the critical Trp382 residue relative to the flavin for efficient vectorial electron transfer leading to photoreduction. The ramifications of vectorial charge redistribution are discussed in the context of the wider class of flavoprotein blue light photoreceptors.
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charge redistribution in oxidized and semiquinone e coli dna photolyase upon photoexcitation stark spectroscopy reveals a rationale for the position of trp382
Journal of the American Chemical Society, 2009Co-Authors: Goutham Kodali, Salim U Siddiqui, Robert J. StanleyAbstract:The electronic structure of the two lowest excited electronic states of FAD and FADHin folate- depleted E. coli DNA photolyase (PLOX and PLSQ, respectively) was measured using absorption Stark spectroscopy. The experimental analysis was supported by TDDFT calculations of both the charge redistribution and the difference dipole moments for the transitions of both oxidation states using lumiflavin as a model. The difference dipole moments and polarizabilities for PLOX are similar to those obtained in our previous work for flavins in simple solvents and in an FMN-containing flavoprotein. No such comparison can be made for PLSQ, as we believe this to be the first experimental report of the direction and magnitude of excited-state charge redistribution in any flavosemiquinone. The picture that emerges from these studies is discussed in the context of electron transfer in photolyase, particularly for the semiquinone photoreduction process, which involves nearby tryptophan residues as electron donors. The direction of charge displacement derived from an analysis of the Stark spectra rationalizes the positioning of the critical Trp382 residue relative to the flavin for efficient vectorial electron transfer leading to photoreduction. The ramifications of vectorial charge redistribution are discussed in the context of the wider class of flavoprotein blue light photoreceptors.
Retsu Miura - One of the best experts on this subject based on the ideXlab platform.
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structure of the transition state analog of medium chain acyl coa dehydrogenase crystallographic and molecular orbital studies on the charge transfer complex of medium chain acyl coa dehydrogenase with 3 thiaoctanoyl coa
Journal of Biochemistry, 2003Co-Authors: Atsuko Satoh, Yoshitaka Nakajima, Ikuko Miyahara, Ken Hirotsu, Takeyuki Tanaka, Yasuzo Nishina, Kiyoshi Shiga, Haruhiko Tamaoki, Chiaki Setoyama, Retsu MiuraAbstract:The flavoenzyme medium-chain acyl-CoA dehydrogenase (MCAD) eliminates the alpha-proton of the substrate analog, 3-thiaoctanoyl-CoA (3S-C8-CoA), to form a charge-transfer complex with deprotonated 3S-C8-CoA. This complex can simulate the metastable reaction intermediate immediately after the alpha-proton elimination of a substrate and before the beta-hydrogen transfer as a hydride, and is therefore regarded as a transition-state analog. The crystalline complex was obtained by co-crystallizing MCAD in the oxidized form with 3S-C8-CoA. The three-dimensional structure of the complex was solved by X-ray crystallography. The deprotonated 3S-C8-CoA was clearly located within the active-site cleft of the enzyme. The arrangement between the flavin ring and deprotonated 3S-C8-CoA is consistent with a charge transfer interaction with the negatively charged acyl-chain of 3S-C8-CoA as an electron donor stacking on the pyrimidine moiety of the flavin ring as an electron acceptor. The structure of the model complex between lumiflavin and the deprotonated ethylthioester of 3-thiabutanoic acid was optimized by molecular orbital calculations. The obtained theoretical structure was essentially the same as that of the corresponding region of the X-ray structure. A considerable amount of negative charge is transferred to the flavin ring system to stabilize the complex by 9.2 kcal/mol. The large stabilization energy by charge transfer probably plays an important role in determining the alignment of the flavin ring with 3S-C8-CoA. The structure of the highest occupied molecular orbital of the complex revealed the electron flow pathway from a substrate to the flavin ring.
Matthew S. Platz - One of the best experts on this subject based on the ideXlab platform.
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the reaction of triplet flavin with indole a study of the cascade of reactive intermediates using density functional theory and time resolved infrared spectroscopy
Journal of the American Chemical Society, 2002Co-Authors: Christopher B. Martin, Meng-lin Tsao, Christopher M. Hadad, Matthew S. PlatzAbstract:As a model for riboflavin, lumiflavin was investigated using density functional theory methods (B3LYP/6-31G* and B3LYP/6-31+G**) with regard to the proposed cascade of intermediates formed after excitation to the triplet state, followed by electron-transfer, proton-transfer, and radical[bond]radical coupling reactions. The excited triplet state of the flavin is predicted to be 42 kcal/mol higher in energy than the singlet ground state, and the pi radical anion lies 45.1 kcal/mol lower in energy than the ground-state flavin and a free electron in the gas phase. The former value compares to a solution-phase triplet energy of 49.8 kcal/mol of riboflavin. For the radical anion, the thermodynamically favored position to accept a proton on the flavin ring system is at N(5). A natural population analysis also provided spin density information for the radicals and insight into the origin of the relative stabilities of the six different calculated hydroflavin radicals. The resulting 5H-LF* radical can then undergo radical[bond]radical coupling reactions, with the most thermodynamically stable adduct being formed at C(4'). Vibrational spectra were also calculated for the transient species. Experimental time-resolved infrared spectroscopic data obtained using riboflavin tetraacetate are in excellent agreement with the calculated spectra for the triplet flavin, the radical anion, and the most stable hydroflavin radical.
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The Photochemistry of Riboflavin Tetraacetate and Nucleosides. A Study Using Density Functional Theory, Laser Flash Photolysis, Fluorescence, UV−Vis, and Time Resolved Infrared Spectroscopy
The Journal of Physical Chemistry B, 2002Co-Authors: Christopher B. Martin, Xiaofeng Shi, Meng-lin Tsao, Dale Karweik, James A. Brooke, Christopher M. Hadad, Matthew S. PlatzAbstract:The photoreaction between riboflavin tetraacetate and nucleosides was investigated using time-resolved infrared spectroscopy (TRIR), laser flash photolysis with UV−vis detection, fluorescence quenching, absorption spectroscopy, and density functional theory calculations. Riboflavin tetraacetate (RBTA) was studied experimentally with indole and with Sheu and Foote's organic soluble silylated guanosine (G‘). Lumiflavin and (R)-2-amino-(S)-4-hydroxy-(R)-5-(hydroxymethyl)-tetrahydrofuran were used as computational models for RBTA and for the sugar moiety of the nucleoside, respectively, using density functional theory calculations (B3LYP/6-31G* and B3LYP/6-31+G**). Vibrational spectra were also calculated for the transient species. Time-resolved infrared spectroscopic data obtained using RBTA are in excellent agreement with the calculated spectra for the triplet flavin, and in the presence of silylated guanosine, with the formation of the most stable hydroflavin radical, RBTH, by an electron transfer−proton t...
