New Insights into the Ultrafast Photophysics of Oxidized and Reduced FAD in Solution

Brazard, Johanna; Usman, Anwar; Lacombat, Fabien; Ley, Christian; Martin, Monique M.; Plaza, Pascal

doi:10.1021/jp110741y

Cited by 34 publications

(70 citation statements)

References 60 publications

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“…S2, and they show a highly multiphasic fluorescence decay, spanning timescales from ∼1 ps to ∼1 ns. However, all decay occurs substantially faster than the intrinsic nanosecond (∼3 ns) decay of FAD (23). The most probable origin of this quenching is electron transfer (ET) from close-by aromatic residues to the excited flavin cofactor.…”

Section: Resultsmentioning

confidence: 99%

Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX

Laptenok

Bouzhir-Sima

Lambry

et al. 2013

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

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In many bacteria the flavoenzyme thymidylate synthase ThyX produces the DNA nucleotide deoxythymidine monophosphate from dUMP, using methylenetetrahydrofolate as carbon donor and NADPH as hydride donor. Because all three substrates bind in close proximity to the catalytic flavin adenine dinucleotide group, substantial flexibility of the ThyX active site has been hypothesized. Using femtosecond time-resolved fluorescence spectroscopy, we have studied the conformational heterogeneity and the conformational interconversion dynamics in real time in ThyX from the hyperthermophilic bacterium Thermotoga maritima. The dynamics of electron transfer to excited flavin adenine dinucleotide from a neighboring tyrosine residue are used as a sensitive probe of the functional dynamics of the active site. The fluorescence decay spanned a full three orders of magnitude, demonstrating a very wide range of conformations. In particular, at physiological temperatures, multiple angstrom cofactor-residue displacements occur on the picoseconds timescale. These experimental findings are supported by molecular dynamics simulations. Binding of the dUMP substrate abolishes this flexibility and stabilizes the active site in a configuration where dUMP closely interacts with the flavin cofactor and very efficiently quenches fluorescence itself. Our results indicate a dynamic selected-fit mechanism where binding of the first substrate dUMP at high temperature stabilizes the enzyme in a configuration favorable for interaction with the second substrate NADPH, and more generally have important implications for the role of active site flexibility in enzymes interacting with multiple poly-atom substrates and products. Moreover, our data provide the basis for exploring the effect of inhibitor molecules on the active site dynamics of ThyX and other multisubstrate flavoenzymes.protein dynamics | flavoprotein | ultrafast fluorescence spectroscopy | quenching C onfigurational flexibility is essential for enzyme function during catalysis. Binding of one or more substrates, accommodation of the transition state where the actual reaction takes place, relaxation to the product state, and release of the product (s) is possible because different configurations of the enzyme are continuously sampled, by thermal or reaction-driven motions, on timescales ranging from femtoseconds to microseconds. The configurational space sampled in a certain time range will depend on the local protein flexibility/energy landscape and the temperature (1). During these configurational changes, distances between constituents of the enzyme complex change. It has been recognized that the fastest (femtosecond to picosecond) localized motions exist alongside the slower motions that occur on the (typically millisecond) timescale of catalysis (2-4).Various experimental techniques allow monitoring changes in interactions resulting from configurational changes. Long-range micro/millisecond domain motions in flexible proteins have been studied by NMR (5) and FRET techniques (6, 7). Molecula...

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Section: Resultsmentioning

confidence: 99%

Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX

Laptenok

Bouzhir-Sima

Lambry

et al. 2013

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

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“…These are characteristic features of excited FAD ox (FAD ox *), already observed in other photolyases or cryptochrome, 25,27,28 different flavoproteins, 42,43 as well as for free FAD ox in solution. 41,44 The evolution of the transient absorption spectra may be divided into three phases, as follows:…”

Section: Spectral Analysismentioning

confidence: 99%

“…In addition, EADS1', as EADS1, is highly characteristic of a bound flavin, its SE band being in particular considerably more blue shifted than that of free FAD. 44 Currently, one may hypothesize the presence of a minority population of MmCPDII that exhibits a larger distance between FAD and the proximal tryptophan. Note that multiexponential FAD ox * decays have repeatedly been observed in BLUF proteins, caused by a FAD-tyrosine electron transfer, and explained in a similar way.…”

Section: Difference Between Eads2 and Eads3mentioning

confidence: 99%

Delocalized hole transport coupled to sub-ns tryptophanyl deprotonation promotes photoreduction of class II photolyases

Lacombat

Espagne

Dozova

et al. 2018

Phys. Chem. Chem. Phys.

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“…It has been demonstrated that FAD in solution can exist in two different conformations [8,9]: open (fluorescent) and stacked (non-fluorescent). Depending on the environment of a particular enzyme, one of these conformations will predominate.…”

Section: -Introductionmentioning

confidence: 99%

Fluorescence of the Flavin group in choline oxidase. Insights and analytical applications for the determination of choline and betaine aldehyde

Ortega

Marcos

Sanz-Vicente

et al. 2016

Talanta

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Choline oxidase (ChOx) is a flavoenzyme catalysing the oxidation of choline (Ch) to betaine aldehyde (BA) and glycine betaine (GB). In this paper a fundamental study of the intrinsic fluorescence properties of ChOx due to Flavin Adenine Dinucleotide (FAD) is presented and some analytical applications are studied in detail. Firstly, an unusual alteration in the excitation spectra, in comparison with the absorption spectra, has been observed as a function of the pH. This is ascribed to a change of polarity in the excited state. Secondly, the evolution of the fluorescence spectra during the reaction seems to indicate that the reaction takes place in two consecutive, but partially overlapped, steps and each of them follows a different mechanism. Thirdly, the chemical system can be used to determine the Ch concentration in the range from 5×10(-6)M to 5×10(-5)M (univariate and multivariate calibration) in the presence of BA as interference, and the joint Ch+BA concentration in the range 5×10(-6)-5×10(-4)M (multivariate calibration) with mean errors under 10%; a semiquantitative determination of the BA concentration can be deduced by difference. Finally, Ch has been successfully determined in an infant milk sample.

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New Insights into the Ultrafast Photophysics of Oxidized and Reduced FAD in Solution

Cited by 34 publications

References 60 publications

Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX

Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX

Delocalized hole transport coupled to sub-ns tryptophanyl deprotonation promotes photoreduction of class II photolyases

Fluorescence of the Flavin group in choline oxidase. Insights and analytical applications for the determination of choline and betaine aldehyde

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