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Robert J Stanley - One of the best experts on this subject based on the ideXlab platform.
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A “How-To” Guide to the Stark Spectroscopy of Flavins and Flavoproteins
Methods of Molecular Biology, 2014Co-Authors: Raymond F. Pauszek, Robert J StanleyAbstract:Flavins and Flavoproteins have been studied by a plethora of spectroscopic techniques. Beginning with the characterization of DNA photolyases and the discovery of the diversity of roles played by excited-state flavins in photobiology, the characterization of the electronic excited state of flavins has become increasingly important. In this protocol, we provide a guide to using Stark spectroscopy in obtaining the degree of electronic charge redistribution in simple flavins and in Flavoproteins. Stark spectroscopy is technically simpler than more common approaches used to explore the structure of the excited state, considerably cheaper to implement, and yet very powerful in its scope. At the end of this guide, we present data taken on non-photobiological Flavoproteins, glutathione reductase and lipoamide dehydrogenase, that suggest that Stark spectroscopy is a unique way to elucidate the electrostatic environment that the flavin cofactor experiences bound inside the protein.
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a how to guide to the stark spectroscopy of flavins and Flavoproteins
Methods of Molecular Biology, 2014Co-Authors: Raymond F. Pauszek, Robert J StanleyAbstract:: Flavins and Flavoproteins have been studied by a plethora of spectroscopic techniques. Beginning with the characterization of DNA photolyases and the discovery of the diversity of roles played by excited-state flavins in photobiology, the characterization of the electronic excited state of flavins has become increasingly important. In this protocol, we provide a guide to using Stark spectroscopy in obtaining the degree of electronic charge redistribution in simple flavins and in Flavoproteins. Stark spectroscopy is technically simpler than more common approaches used to explore the structure of the excited state, considerably cheaper to implement, and yet very powerful in its scope. At the end of this guide, we present data taken on non-photobiological Flavoproteins, glutathione reductase and lipoamide dehydrogenase, that suggest that Stark spectroscopy is a unique way to elucidate the electrostatic environment that the flavin cofactor experiences bound inside the protein.
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Advances in flavin and flavoprotein optical spectroscopy.
Antioxidants & Redox Signaling, 2001Co-Authors: Robert J StanleyAbstract:Flavins and Flavoproteins are versatile redox cofactors that can perform both one- and two-electron transfer. Because they are highly colored in all three oxidation states, optical spectroscopy has been exploited for decades to study these redox changes. This review summarizes the application of optical spectroscopies to flavins and Flavoproteins since 1990. Special emphasis is placed on new techniques, such as Stark spectroscopy, as well as significant refinements in more well known techniques, such as resonance Raman spectroscopy and ultrafast spectroscopy.
Vincent Massey - One of the best experts on this subject based on the ideXlab platform.
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[44] Reversible resolution of Flavoproteins into apoproteins and free flavins
Methods in Enzymology, 2004Co-Authors: Mazhar Husain, Vincent MasseyAbstract:Publisher Summary This chapter describes the reversible resolution of Flavoproteins into apoproteins and free flavins. Flavoproteins exhibit a great degree of variation in their ease of resolution into flavin and apoenzyme. With some of them, partial resolution is obtained under very mild conditions, such as dialysis against water or buffers. Simple dialysis or gel filtration, therefore, cannot be expected on theoretical grounds to yield pure apoprotein, because as the free apoprotein accumulates, the rate of reassociation, being a second order reaction, becomes competitive with the physical removal of the flavin by dialysis or gel filtration. Some Flavoproteins can be resolved satisfactorily by a number of methods, while others require rather special treatment. With other Flavoproteins, however, the mechanism of reconstitution may be quite complex, with the formation of active enzyme requiring minutes or even hours depending on the temperature. As most apoproteins are less stable than their corresponding holoproteins, the apoprotein and flavin are first incubated on ice for some time. A multiphasic reactivation process may be applied if the expected activity is not regained.
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syntheses and applications of flavin analogs as active site probes for Flavoproteins
Methods in Enzymology, 1997Co-Authors: Yerramilli V S N Murthy, Vincent MasseyAbstract:Publisher Summary This chapter discusses the synthesis of a range of new flavins and their potentials as active-site probes for Flavoproteins. A few new flavins with substitutions such as trifluoromethyl, chloro, azido, carbonyl, and mercapto at the N-5 position are successfully synthesized. The selective introduction of fluorine into biologically interesting molecules is fast emerging as an effective tool because of the unique properties of this halogen. Because fluorine is smallest next to hydrogen, its substitution often results in minimal steric constraints. Also, because fluorine can act as hydrogen bond acceptor, the replacement of hydroxyl with fluorine allows the molecule to retain its properties. The N-10 ribityl side chain of flavins is generally considered as only a binding anchor in Flavoproteins, with no positive role in catalysis. However, the X-ray crystal structures of various Flavoproteins such as Old Yellow Enzyme, acyl-CoA dehydrogenase, glutathione reductase, and lipoamide dehydrogenase show involvement of ribitylhydroxylgroups in hydrogen bonds that can regulate catalytically important amino acid residues.
Milagros Medina - One of the best experts on this subject based on the ideXlab platform.
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ENDOR and related EMR methods applied to flavoprotein radicals
Applied Magnetic Resonance, 2007Co-Authors: Milagros Medina, Richard CammackAbstract:Flavoproteins are involved in a wide range of biological processes, owing to the versatility of the isoalloxazine moiety of flavin, which can undergo both one- and two-electron reactions with the formation of three oxidation states. Paramagnetic semiquinone radical states are stabilised in some Flavoproteins and appear as transient intermediates in the reaction of many others. The apoprotein controls the reactivity of flavin semiquinones, including redox potentials, protonation states and access to substrates. Most Flavoproteins are involved in oxidation-reduction processes, but some catalyze different types of reactions involving radical intermediates. Anionic and neutral flavin radicals are found in Flavoproteins and are distinguished by their line widths in X-band electron paramagnetic resonance. Electron-nuclear double resonance, electron spin echo envelope modulation and hyperfine sublevel correlation spectroscopy make it possible to observe biological electron transfer and catalysis at the level of the electronic structure of the intermediate states. They provide information about the protein environment of flavin semiquinone radicals and their interactions with nearby nuclear and electron spins. Hyperfine couplings, particularly to the 8-methyl protons on the flavin ring, are a sensitive probe of perturbations of the flavin environment. They demonstrate differences in polarity of the flavin binding site and changes that occur in flavoenzymes during binding of substrates.
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one and two dimensional eseem spectroscopy of Flavoproteins
Biochemistry, 1997Co-Authors: Jesus I Martinez, Pablo J Alonso, Carlos Gomezmoreno, Milagros MedinaAbstract:One- and two-dimensional (1D and 2D) electron spin echo envelope modulation (ESEEM) spectroscopy was applied to study the flavin cofactors in the neutral semiquinone states of flavodoxin and ferredoxin-NADP + reductase (FNR) from the cyanobacterium Anabaena PCC 7119, and the anionic semiquinone state of cholesterol oxidase from BreVibacterium sterolicum. High-resolution crystal structures are available for all these proteins. Three- and 4-pulse ESEEM and hyperfine sublevel correlation spectroscopy (HYSCORE) techniques at X-band were used. HYSCORE spectra showed correlations between transitions caused by interaction of the isoalloxazine unpaired electronic spin present in the semiquinone state with several nitrogen and hydrogen nuclei. Measurements of isotopic labeled samples (( 15 N)FMN flavodoxin and ( 2 H)flavodoxin) allowed the assignment of all the detected transitions to nuclei belonging to the FMN cofactor group. Interactions of nitrogens in positions 1 and 3 of the isoalloxazine ring were determined to have isotropic hyperfine coupling constants in the 1-2 and 0.5-1 MHz ranges for all the different flavoprotein semiquinones studied. Information about the quadrupolar term of these nuclei was also obtained. An intense correlation in the negative quadrant was detected. It has been associated to the strongly interacting N(10) nucleus. The complete hyperfine term parameters (including the sign) were obtained from detailed analysis of this signal, being the quadrupolar parameter, K, also estimated. Another correlation in the HYSCORE spectra, corresponding to hydrogen bound to the N(5) position in neutral flavin semiquinones, was detected. Its interaction parameters were also determined. This study demonstrates that ESEEM spectroscopy, and in particular the HYSCORE technique, are of particular utility for detecting and assigning nuclear transition frequencies in flavoprotein semiquinones. Moreover, the results reported here are complementary to ENDOR studies, and both techniques together provide an important tool for obtaining information about spin distribution in the flavin ring of Flavoproteins in the semiquinone state.
Frank E. Frerman - One of the best experts on this subject based on the ideXlab platform.
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31p nmr spectroscopy of human and paracoccus denitrificans electron transfer Flavoproteins and 13c and 15n nmr spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states
FEBS Journal, 1998Co-Authors: Kurt J. Griffin, Franz Muller, Gregory D. Degala, Wolfgang Eisenreich, Adelbert Bacher, Frank E. FrermanAbstract:Human and Paracoccus denitrificans wild-type electron transfer Flavoproteins have been investigated by 31P-NMR in the oxidised and reduced states. The 31P chemical shifts of the diphosphate moiety of the protein-bound FAD were similar in the proteins and were independent of the redox state. The chemical shifts were remarkably similar to those of ferredoxin−NADP+ reductase and, to a lesser degree, with those of NADPH−cytochrome P-450 reductase. The wild-type human electron transfer apoprotein was reconstituted with [2,4a-13C2]FAD, [4,10a-13C2]FAD, or [U-15N4]FAD. The reconstituted proteins were studied by 13C- and 15N-NMR techniques in the oxidised and reduced states. The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of Flavoproteins published in the literature. In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD. The N(1) atom is also hydrogen bonded and, as shown by X-ray data, involves the C′(4)-OH group of FAD. The sp2 hybridisation of N(10) is small compared to other Flavoproteins. In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD. The N(1) atom is ionised as observed also in other Flavoproteins when investigated by NMR. The intramolecular hydrogen bond between the C′(4)-OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein-bound FAD in both redox states. The N(10) atom in the reduced protein is highly sp3 hybridised in comparison to those of other Flavoproteins.
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31P‐NMR spectroscopy of human and Paracoccus denitrificans electron transfer Flavoproteins, and 13C‐ and 15N‐NMR spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states
FEBS Journal, 1998Co-Authors: Kurt J. Griffin, Franz Muller, Gregory D. Degala, Wolfgang Eisenreich, Adelbert Bacher, Frank E. FrermanAbstract:Human and Paracoccus denitrificans wild-type electron transfer Flavoproteins have been investigated by 31P-NMR in the oxidised and reduced states. The 31P chemical shifts of the diphosphate moiety of the protein-bound FAD were similar in the proteins and were independent of the redox state. The chemical shifts were remarkably similar to those of ferredoxin−NADP+ reductase and, to a lesser degree, with those of NADPH−cytochrome P-450 reductase. The wild-type human electron transfer apoprotein was reconstituted with [2,4a-13C2]FAD, [4,10a-13C2]FAD, or [U-15N4]FAD. The reconstituted proteins were studied by 13C- and 15N-NMR techniques in the oxidised and reduced states. The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of Flavoproteins published in the literature. In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD. The N(1) atom is also hydrogen bonded and, as shown by X-ray data, involves the C′(4)-OH group of FAD. The sp2 hybridisation of N(10) is small compared to other Flavoproteins. In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD. The N(1) atom is ionised as observed also in other Flavoproteins when investigated by NMR. The intramolecular hydrogen bond between the C′(4)-OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein-bound FAD in both redox states. The N(10) atom in the reduced protein is highly sp3 hybridised in comparison to those of other Flavoproteins.
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Three-dimensional structure of human electron transfer flavoprotein to 2.1-A resolution.
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: David L. Roberts, Frank E. FrermanAbstract:Mammalian electron transfer Flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-A resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the α subunit and the third domain is made up entirely by the β subunit. The N-terminal portion of the α subunit and the majority of the β subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the α subunit. Alignment of all the known sequences for the ETF α subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of αT266, suggesting why the pathogenic mutation, αT266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4′-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between αH286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.
Franz Muller - One of the best experts on this subject based on the ideXlab platform.
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Methods used to resolve reversible Flavoproteins into the constituents apoflavoprotein and prosthetic group
Chemistry and Biochemistry of Flavoenzymes, 2018Co-Authors: Franz Muller, W.j.h. Van BerkelAbstract:There are two classes of Flavoproteins with respect to the interaction between the apoflavoprotein and its prosthetic group. In one class, the flavin is often tightly, but noncovalently bound. In the other class, the interaction between the two components is of covalent nature, the flavin being attached to an amino acid residue. This chapter discusses the most commonly used procedures for the preparation of apoFlavoproteins. Although the development of methods for the reversible resolution of Flavoproteins can be tedious and time consuming, especially when the goal is the achievement of a high reconstitutability, some studies should be included in the basic work done to characterize a new flavoprotein. Although the covalent chromatography method could be applied to any flavoprotein possessing an easily accessible sulfhydryl group, the method probably does not always give satisfactory results. The acid ammonium sulfate treatment is a rapid method to probe the possible preparation and stability/reconstitutability of apoFlavoproteins on an analytical scale.
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nmr spectroscopy on flavins and Flavoproteins
Methods of Molecular Biology, 2014Co-Authors: Franz MullerAbstract:: (1)H-, (11)B-, (13)C-, (15)N-, (17)O-, (19)F-, and (31)P-NMR chemical shifts of flavocoenzymes and derivatives of it, as well as of alloxazines and isoalloxazinium salts, from NMR experiments performed under various experimental conditions (e.g., dependence of the chemical shifts on temperature, concentration, solvent polarity, and pH) are reported. Also solid-state (13)C- and (15)N-NMR experiments are described revealing the anisotropic values of corresponding chemical shifts. These data, in combination with a number of coupling constants, led to a detailed description of the electronic structure of oxidized and reduced flavins. The data also demonstrate that the structure of oxidized flavin can assume a configuration deviating from coplanarity, depending on substitutions in the isoalloxazine ring, while that of reduced flavin exhibits several configurations, from almost planar to quite bended. The complexes formed between oxidized flavin and metal ions or organic molecules revealed three coordination sites with metal ions (depending on the chemical nature of the ion), and specific interactions between the pyrimidine moiety of flavin and organic molecules, mimicking specific interactions between apoFlavoproteins and their coenzymes. Most NMR studies on Flavoproteins were performed using (13)C- and (15)N-substituted coenzymes, either specifically enriched in the pterin moiety of flavin or uniformly labeled flavins. The chemical shifts of free flavins are used as a guide in the interpretation of the chemical shifts observed in Flavoproteins. Although the hydrogen-bonding pattern in oxidized and reduced Flavoproteins varies considerably, no correlation is obvious between these patterns and the corresponding redox potentials. In all reduced Flavoproteins the N(1)H group of the flavocoenzyme is deprotonated, an exception is thioredoxin reductase. Three-dimensional structures of only a few Flavoproteins, mostly belonging to the family of flavodoxins, have been solved. Also the kinetics of unfolding and refolding of flavodoxins has been investigated by NMR techniques. In addition, (31)P-NMR data of all so far studied Flavoproteins and some (19)F-NMR spectra are discussed.
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31P‐NMR spectroscopy of human and Paracoccus denitrificans electron transfer Flavoproteins, and 13C‐ and 15N‐NMR spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states
FEBS Journal, 1998Co-Authors: Kurt J. Griffin, Franz Muller, Gregory D. Degala, Wolfgang Eisenreich, Adelbert Bacher, Frank E. FrermanAbstract:Human and Paracoccus denitrificans wild-type electron transfer Flavoproteins have been investigated by 31P-NMR in the oxidised and reduced states. The 31P chemical shifts of the diphosphate moiety of the protein-bound FAD were similar in the proteins and were independent of the redox state. The chemical shifts were remarkably similar to those of ferredoxin−NADP+ reductase and, to a lesser degree, with those of NADPH−cytochrome P-450 reductase. The wild-type human electron transfer apoprotein was reconstituted with [2,4a-13C2]FAD, [4,10a-13C2]FAD, or [U-15N4]FAD. The reconstituted proteins were studied by 13C- and 15N-NMR techniques in the oxidised and reduced states. The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of Flavoproteins published in the literature. In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD. The N(1) atom is also hydrogen bonded and, as shown by X-ray data, involves the C′(4)-OH group of FAD. The sp2 hybridisation of N(10) is small compared to other Flavoproteins. In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD. The N(1) atom is ionised as observed also in other Flavoproteins when investigated by NMR. The intramolecular hydrogen bond between the C′(4)-OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein-bound FAD in both redox states. The N(10) atom in the reduced protein is highly sp3 hybridised in comparison to those of other Flavoproteins.
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31p nmr spectroscopy of human and paracoccus denitrificans electron transfer Flavoproteins and 13c and 15n nmr spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states
FEBS Journal, 1998Co-Authors: Kurt J. Griffin, Franz Muller, Gregory D. Degala, Wolfgang Eisenreich, Adelbert Bacher, Frank E. FrermanAbstract:Human and Paracoccus denitrificans wild-type electron transfer Flavoproteins have been investigated by 31P-NMR in the oxidised and reduced states. The 31P chemical shifts of the diphosphate moiety of the protein-bound FAD were similar in the proteins and were independent of the redox state. The chemical shifts were remarkably similar to those of ferredoxin−NADP+ reductase and, to a lesser degree, with those of NADPH−cytochrome P-450 reductase. The wild-type human electron transfer apoprotein was reconstituted with [2,4a-13C2]FAD, [4,10a-13C2]FAD, or [U-15N4]FAD. The reconstituted proteins were studied by 13C- and 15N-NMR techniques in the oxidised and reduced states. The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of Flavoproteins published in the literature. In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD. The N(1) atom is also hydrogen bonded and, as shown by X-ray data, involves the C′(4)-OH group of FAD. The sp2 hybridisation of N(10) is small compared to other Flavoproteins. In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD. The N(1) atom is ionised as observed also in other Flavoproteins when investigated by NMR. The intramolecular hydrogen bond between the C′(4)-OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein-bound FAD in both redox states. The N(10) atom in the reduced protein is highly sp3 hybridised in comparison to those of other Flavoproteins.