New flavins for old: artificial flavins as active site probes of flavoproteins

Ghisla, Sandro; Massey, Vincent

doi:10.1042/bj2390001

Cited by 195 publications

(153 citation statements)

References 78 publications

(75 reference statements)

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“…Interestingly many flavoenzymes can reversibly be converted to their corresponding apoproteins under relatively mild conditions [33], and subsequently be reconstituted by incubation with an excess of FAD [34]. This property may be important for the interpretation of experiments of our studies describing the epoxidation of xanthophylls in the thylakoid membrane of spinach.…”

Section: Discussionmentioning

confidence: 92%

FAD is a further essential cofactor of the NAD(P)H and O₂‐dependent zeaxanthin‐epoxidase

1995

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dase was suggested to be associated with the stromal side of this membrane [16]. Recently, Zea epoxidation activity was attributed to isolated LHCsII [19,20].In this study we demonstrate that a further cofactor, flavin adenine dinucleotide (FAD), is involved in the NAD(P)H-and O2-dependent epoxidation of Zea. Presumable, one part of the protein-associated flavin moiety is lost during the isolation procedure of the thylakoid membranes and consequently the epoxidase activity is attenuated. Addition of FAD to the thylakoid suspensions restores the enzyme activity. A further indication for the importance of this flavin during the Zea epoxidation is the strong effect of DPI, an inhibitor of flavin-containing enzymes [21,22]. Materials and methods Plant materialSpinach plants (Spinacia oleracea L. cv. Butterfly) were cultivated in a growth chamber (10 h light, 23°C, 55% rel. humidity; 14 h dark, 15°C, 70% rel. humidity). The illumination period was started and terminated by a dawn and dusk period of 1 h. For illumination HQI-R 250 W bulbs (Osram) were used (140 pmol photons-m -2-s-%. All experiments were performed with 6-week-old leaves.

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Section: Discussionmentioning

confidence: 92%

FAD is a further essential cofactor of the NAD(P)H and O₂‐dependent zeaxanthin‐epoxidase

1995

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“…Comparison of the reactivity of GTN with reduced flavins and reduced deazaflavins does, however, give support to the radical pathway of Scheme 1B. Because of the thermodynamic properties of 5-deazaflavins (26), with redox potentials of ϷϪ650 mV for the oxidized/semiquinone (ox͞sq) couple, ϷϪ300 mV for the ox͞red couple, and Ϸϩ50 mV for the sq͞red couple, 5-deazaflavins generally are considered as incompetent in radical reaction pathways (30,31). On the other hand, they are quite competent for hydride transfer, and in the case of acyl CoA dehydrogenase, the reduced 5-deazaFAD enzyme form was found to be oxidized by crotonyl CoA some 4 times faster than normal enzyme (32).…”

Section: Discussionmentioning

confidence: 99%

Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

Meah

Brown

Chakraborty

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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The reaction of the old yellow enzyme and reduced flavins with organic nitrate esters has been studied. Reduced flavins have been found to react readily with glycerin trinitrate (GTN ) (nitroglycerin) and propylene dinitrate, with rate constants at pH 7.0, 25°C of 145 M ؊1 s ؊1 and 5.8 M ؊1 s ؊1 , respectively. With GTN, the secondary nitrate was removed reductively 6 times faster than the primary nitrate, with liberation of nitrite. With propylene dinitrate, on the other hand, the primary nitrate residue was 3 times more reactive than the secondary residue. In the old yellow enzyme-catalyzed NADPH-dependent reduction of GTN and propylene dinitrate, ping-pong kinetics are displayed, as found for all other substrates of the enzyme. Rapid-reaction studies of mixing reduced enzyme with the nitrate esters show that a reduced enzyme-substrate complex is formed before oxidation of the reduced flavin. The rate constants for these reactions and the apparent Kd values of the enzyme-substrate complexes have been determined and reveal that the rate-limiting step in catalysis is reduction of the enzyme by NADPH. Analysis of the products reveal that with the enzymecatalyzed reactions, reduction of the primary nitrate in both GTN and propylene dinitrate is favored by comparison with the freeflavin reactions. This preferential positional reactivity can be rationalized by modeling of the substrates into the known crystal structure of the enzyme. In contrast to the facile reaction of free reduced flavins with GTN, reduced 5-deazaflavins have been found to react some 4 -5 orders of magnitude slower. This finding implies that the chemical mechanism of the reaction is one involving radical transfers.

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“…This conclusion is based upon halide elimination from ␤-substituted substrates (5), mechanism-based inactivation by propargyllic substrates (6), and kinetic isotope effects (7). The substrate carbanion is proposed to form an adduct at the N(5) position of flavin; such an adduct would then break down to form the reduced flavin and keto or imino acid product (8). Evidence for such an adduct comes from: (a) the trapping of a species with the properties of an N(5) adduct when glycolate is a substrate for lactate oxidase (9) and (b) the formation of adducts when nitromethane or nitroethane carbanion is used as substrate for D-amino acid oxidase (10).…”

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confidence: 99%

Identification of the Naturally Occurring Flavin of Nitroalkane Oxidase from Fusarium oxysporum as a 5-Nitrobutyl-FAD and Conversion of the Enzyme to the Active FAD-containing Form

Gadda

Edmondson

Russell

et al. 1997

Journal of Biological Chemistry

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Nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to aldehydes with production of nitrite and hydrogen peroxide. The UVvisible absorbance spectrum of the purified enzyme shows a single absorption peak at 336 nm with an extinction coefficient of 7.4 mM ؊1 cm ؊1 . Upon denaturation of the enzyme at pH 7.0, a stoichiometric amount of FAD is released. The spectral properties of the enzyme as isolated are consistent with an N(5) adduct of the flavin. This is not due to a covalent linkage with the protein, since the free flavin adduct can be isolated from the enzyme at pH 2.1. The free flavin adduct shows an absorbance spectrum with a max at 346 nm (10.7 mM ؊1 cm ؊1 ) and is not fluorescent. Under alkaline conditions the free adduct decays, yielding FAD; the rate of this process is pH-dependent with a pK a of 7.4. Adduct decay is also observed with the native enzyme; in this case, however, the rate of decay is 160-fold slower (at pH 8.0) and not dependent on pH. During this process a large increase in enzymatic activity (ϳ26-fold at pH 7.0) is observed, the rate of which is equal to the rate of flavin adduct conversion to FAD. Thus, the native flavin adduct is not active but can be converted to FAD, the active form of the flavin. Maximal activation is pH-and FAD-dependent; two groups with pK a values of 5.65 ؎ 0.25 and 8.75 ؎ 0.05 must be unprotonated and protonated, respectively. The m/z ؊ of the free flavin adduct is 103.0645 higher than that of FAD, as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This corresponds to a molecule of nitrobutane linked to FAD. A mechanism is proposed for the formation in vivo of the nitrobutyl-FAD of nitroalkane oxidase.Flavoprotein oxidases comprise a large group of enzymes that catalyze the removal of a hydride equivalent from a substrate, transferring the electrons initially to the flavin cofactor and then to molecular oxygen to form hydrogen peroxide and the oxidized product. The most studied examples are the ␣-hydroxy acid oxidases, such as lactate oxidase (1), glycolate oxidase (2), and flavocytochrome b 2 (3) and the amino acid oxidases, of which D-amino acid oxidase is the best understood (4). There is strong evidence that the first step in the formation of the respective keto or imino acid products is removal of the substrate ␣-proton to form a carbanion. This conclusion is based upon halide elimination from ␤-substituted substrates (5), mechanism-based inactivation by propargyllic substrates (6), and kinetic isotope effects (7). The substrate carbanion is proposed to form an adduct at the N(5) position of flavin; such an adduct would then break down to form the reduced flavin and keto or imino acid product (8). Evidence for such an adduct comes from: (a) the trapping of a species with the properties of an N(5) adduct when glycolate is a substrate for lactate oxidase (9) and (b) the formation of adducts when nitromethane or nitroethane carbanion is used as substrate for D-amino acid oxidase (10). ...

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New flavins for old: artificial flavins as active site probes of flavoproteins

Cited by 195 publications

References 78 publications

FAD is a further essential cofactor of the NAD(P)H and O₂‐dependent zeaxanthin‐epoxidase

FAD is a further essential cofactor of the NAD(P)H and O₂‐dependent zeaxanthin‐epoxidase

Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

Identification of the Naturally Occurring Flavin of Nitroalkane Oxidase from Fusarium oxysporum as a 5-Nitrobutyl-FAD and Conversion of the Enzyme to the Active FAD-containing Form

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New flavins for old: artificial flavins as active site probes of flavoproteins

Cited by 195 publications

References 78 publications

FAD is a further essential cofactor of the NAD(P)H and O2‐dependent zeaxanthin‐epoxidase

FAD is a further essential cofactor of the NAD(P)H and O2‐dependent zeaxanthin‐epoxidase

Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

Identification of the Naturally Occurring Flavin of Nitroalkane Oxidase from Fusarium oxysporum as a 5-Nitrobutyl-FAD and Conversion of the Enzyme to the Active FAD-containing Form

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FAD is a further essential cofactor of the NAD(P)H and O₂‐dependent zeaxanthin‐epoxidase

FAD is a further essential cofactor of the NAD(P)H and O₂‐dependent zeaxanthin‐epoxidase