Mapping Solvation Dynamics at the Function Site of Flavodoxin in Three Redox States

Chang, Chih Wei; He, Ting; Guo, Lijun; Stevens, Jeffrey A.; Li, Tanping; Wang, Limin; Zhong, Dongping

doi:10.1021/ja1050154

Cited by 50 publications

(75 citation statements)

References 54 publications

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“…We have measured the active-site solvation dynamics in the reduced FADH -state and have found that the relaxation takes place on the timescales from a few picoseconds to subnanoseconds (21). The similar timescales would be expected for the oxidized FAD state (22) and therefore the ET dynamics shows a stretched behavior, Ae −ðt=τÞ β , where A is the amplitude, τ is the lifetime, and β here is a stretched parameter. Using < τ > = τ β Γ 1 β , we obtained the average lifetimes of the forward ET in 19 ps and the back ET in 100 ps (Fig.…”

Section: Identifying New Electron Donors (Ade and W384) In Initial Elmentioning

confidence: 65%

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

Liu¹,

Tan

Guo³

et al. 2013

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

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170

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The flavin cofactor in photoenzyme photolyase and photoreceptor cryptochrome may exist in an oxidized state and should be converted into reduced state(s) for biological functions. Such redox changes can be efficiently achieved by photoinduced electron transfer (ET) through a series of aromatic residues in the enzyme. Here, we report our complete characterization of photoreduction dynamics of photolyase with femtosecond resolution. With various site-directed mutations, we identified all possible electron donors in the enzyme and determined their ET timescales. The excited cofactor behaves as an electron sink to draw electron flow from a series of encircling aromatic molecules in three distinct layers from the active site in the center to the protein surface. The dominant electron flow follows the conserved tryptophan triad in a hopping pathway across the layers with multiple tunneling steps. These ET dynamics occur ultrafast in less than 150 ps and are strongly coupled with local protein and solvent relaxations. The reverse electron flow from the flavin is slow and in the nanosecond range to ensure high reduction efficiency. With 12 experimentally determined elementary ET steps and 6 ET reaction pairs, the enzyme exhibits a distinct reduction-potential gradient along the same aromatic residues with favorable reorganization energies to drive a highly unidirectional electron flow toward the active-site center from the protein surface.protein electron transfer | flavin photoreduction | femtosecond dynamics | electron flow directionality | reduction potential funnel P hotolyase and cryptochrome are evolutionally related and contain a flavin adenine dinucleotide (FAD) as the catalytic cofactor with a unique bent structure in the active sites, but the two perform different functions: photolyase repairs UV-damaged DNA and cryptochrome functions as a photoreceptor for regulation of plant growth or synchronization of circadian rhythm (1-5). The active state of the cofactor in vivo is in the anionic hydroquinone form (FADH -) in photolyase (6), but currently the redox status of flavin in cryptochrome is under debate with some studies suggesting flavin to be in oxidized (FAD), whereas others claiming anionic (FAD -/FADH -) states for the functional form in vivo (7-10). However, in vitro, the cofactor is oxidized to FAD and/or FADH • in photolyase but only appears in the oxidized FAD state in cryptochrome. Thus, it appears that reduction of the oxidized FAD is necessary for its transformation into the active state. Photoinduced electron transfer (ET) is an effective way for such redox state changes and it is conceivable that the photoreduction of FAD could be a primary process for signal initiation in cryptochrome (11).In this study, we used Escherichia coli photolyase (EcPL) as a model system for systematic studies of electron flow into the excited cofactor FAD. As shown in Fig. 1, more than 10 aromatic acid residues (W and Y) encircle the flavin cofactor around the active site (12). These aromatic residues not only are cri...

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Section: Identifying New Electron Donors (Ade and W384) In Initial Elmentioning

confidence: 65%

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

Liu¹,

Tan

Guo³

et al. 2013

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

Self Cite

170

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“…[3] We calculated vibrationally-resolved absorption spectra of the three redox states of FMN and obtained excellent agreement with the experimental spectra. However, our results indicate that specific short-range interactions play a negligible role in the determination of the optical spectra of FMN in its three different redox states, while the intrinsic properties play an important role.…”

mentioning

confidence: 56%

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mentioning

confidence: 99%

Intrinsic Property of Flavin Mononucleotide Controls its Optical Spectra in Three Redox States

Ai¹,

Tian²,

Liao³

et al. 2011

ChemPhysChem

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Optical spectroscopic techniques have been the primary tools in detecting peptide conformation dynamics, [1] protein folding, [2] local solvation dynamics, [3] electron transfer processes, [4] and photochemical reactions. [5] The spectra reflect molecular properties in both internal (e.g. molecular structure and charge distribution) and external (environmental) aspects. In particular, the environmental influence can be classified into implicit and specific interactions between the system and the surrounding protein or solvents. These long-range and short-range interactions play an important role in biological molecules, also regulating their biological functions and activities. Revealing the connection of internal and external properties and their roles in the biological reactions has become the most challenging task for spectroscopic experimentalists because of the complexity and the dynamics of the external environment. There is an obvious gap between actual spectral information and possible interpretations, which could only be filled by theoretical modeling, which is capable of providing fine structures of spectral profiles, such as vibrational progressions.Herein, we use flavin mononucleotide (FMN) as a model system to demonstrate the importance of theoretical modeling and its power to reveal details that cannot be accessed directly by measurements.Flavodoxin as a flavoprotein is much involved in a variety of biochemical and physiological functions, such as interchangeability with ferredoxins, carbon dioxide and nitrogen fixation, oxygen activation, and so forth.[6] Its practical applications in the design of protein/metal nanostructures or drugs have drawn much attention recently.[6b] Flavodoxin contains FMN as a cofactor, which is non-covalently bonded to flavodoxin, expressing its redox activity through electron transport between different oxidation states. This intrinsic property was suggested to be tailored by the host apoprotein.[6a] Due to its important role in biological functions and its unique redox properties, FMN has been extensively studied by experimentalists and theoreticians.[6b] Recently, Chang et al. characterized the different ultrafast solvation dynamics of the three redox states (the oxidized form FMN, semiquinone FMNHC and hydroquinone FMNH À , Figure 1 a) by ultrafast femtosecond measurements, which are suggested to arise from the local protein plasticity and water network flexibility. [3] We calculated vibrationally-resolved absorption spectra of the three redox states of FMN and obtained excellent agreement with the experimental spectra. However, our results indicate that specific short-range interactions play a negligible role in the determination of the optical spectra of FMN in its three different redox states, while the intrinsic properties play an important role.The initial geometries were extracted from X-ray crystal structures of Desulfovibrio vulgaris flavodoxins in the three different redox states [7] (PDB codes:2FX3, 2FX4, 2FX5), with manually added hydrogens. The models wit...

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“…S3 A and B). This analysis indicates that red shifts occur concomitant with fluorescence decay on the timescale <200 ps, and presumably reflect charge relaxation processes of the flavin environment (25). The nonexponentiality of the decay can be assigned to a distribution of conformations of the donor-acceptor pairs that do not or only partly interconvert during the excited state lifetime.…”

Section: Resultsmentioning

confidence: 87%

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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Mapping Solvation Dynamics at the Function Site of Flavodoxin in Three Redox States

Cited by 50 publications

References 54 publications

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

Intrinsic Property of Flavin Mononucleotide Controls its Optical Spectra in Three Redox States

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

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