Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii. A multidimensional time-resolved polarized fluorescence study

Bastiaens, Philippe I. H.; Hoek, A. van; Benen, J.A.E.; Brochon, Jean‐Claude; Visser, Antonie J. W. G.

doi:10.1016/s0006-3495(92)81659-4

Cited by 64 publications

(48 citation statements)

References 38 publications

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“…We note that temperature-dependent fluorescence decay times have been reported for the flavoprotein lipoamide dehydrogenase (29). The FAD cofactors in this protein have an extremely high fluorescence quantum yield and are not quenched; here, the activation of the fluorescence decay was due to distinct conformational changes that are static on the nanosecond timescale (29). The analysis in the present work by contrast highlights dynamic conformational changes occurring on the picosecond timescale.…”

Section: Discussionsupporting

confidence: 45%

“…For these reasons we interpret the temperature dependence observed in this work as originating from changes in the distance-dependent electronic coupling between the reactants, rather than from activation barriers. We note that temperature-dependent fluorescence decay times have been reported for the flavoprotein lipoamide dehydrogenase (29). The FAD cofactors in this protein have an extremely high fluorescence quantum yield and are not quenched; here, the activation of the fluorescence decay was due to distinct conformational changes that are static on the nanosecond timescale (29).…”

Section: Discussionmentioning

confidence: 53%

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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: Discussionsupporting

confidence: 45%

Section: Discussionmentioning

confidence: 53%

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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“…Integration of the amplitudes, p(4), yields initial anisotropies of 0.35 for both the FMN and flavodoxin. For other flavoproteins identical initial anisotropies have been demonstrated (Bastiaens et al, 1992b).…”

Section: Time-resolved Fluorescence Anisotropymentioning

confidence: 75%

Flavin dynamics in oxidized Clostridium beijerinckii flavodoxin as assessed by time‐resolved polarized fluorescence

Leenders

Hoek

Iersel

et al. 1993

European Journal of Biochemistry

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The time-resolved fluorescence characteristics of flavin in oxidized flavodoxin isolated from the anaerobic bacterium Clostridium beijerinckii have been examined. The fluorescence intensity decays were analyzed using the maximum-entropy method. It is demonstrated that there exist large differences in fluorescence behaviour between free and protein-bound FMN. Three fluorescence lifetime components are found in oxidized flavodoxin, two of which are not present in the fluorescenceintensity decay of free FMN. The main component is distributed at 30 ps, with relative contribution of 90%. Another minor component (4% contribution) is distributed at 0.5 ns. The third component is distributed at 4.8 ns (6%), coinciding with the main distribution present in the fluorescence decay of free FMN. The results allowed us to determine the dissociation constant, Kd = 2.61 X 10--'"M (at 20°C).Collisional fluorescence-quenching experiments revealed that the flavin moiety responsible for the longest fluorescence lifetime is, at least partially, exposed to the solvent. The shortest lifetime is not affected significantly, indicating that it possibly originates from an active-site conformation in which the flavin is more or less buried in the protein and not accessible to iodide.The fluorescence anisotropy behaviour of free and protein-bound FMN was examined and analyzed with the maximum-entropy method. It was found that an excess of apoflavodoxin is required to detect differences between free and protein-bound FMN. In free FMN one single distribution of rotational correlation times is detected, whereas in flavodoxin the anisotropy decay is composed of more than one distribution.Associative analysis of fluorescence anisotropy decays shows that part of the 4.8 ns fluorescence lifetime present in the flavodoxin fluorescence decay, is coupled to a rotational correlation time similar to that of free FMN in solution, while another part of this lifetime is coupled to a longer correlation time of about 1 ns. This finding is in accordance with earlier studies [Barman, B. G. & Tollin, G. (1972) Biochemistry 11, 4746-47541 in which it was proposed that the first binding step of the flavin to the protein involves the phosphate group rather than another part of the FMN.The two shortest fluorescence lifetimes, which do not carry information on the long-term rotational behaviour of the protein, seem nonetheless to be associated with a longer rotational correlation time which is comparable to overall protein tumbling. These lifetime components probably originate from a complex in which the flavin-ring system is more or less immobilized within the protein matrix.Flavodoxins are low-molecular-mass electron-transport proteins (14-25 kDa) which can substitute for ferredoxin in a number of reactions (Mayhew, 1971 ;Mayhew and Ludwig, 1975;Mayhew and Tollin, 1992). Flavodoxins can be isolated from some algae and several anaerobic and aerobic bacteria grown under iron-containing or iron-deficient conditions (Knight and Hardy, 1966;Benemann et al., 1969;Mayh...

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“…For experimental decay, 4096 channels were used with a time spacing of 8.3 ps per channel. Erythrosine B in nanopure water (with a known single lifetime of 80 ps) served as a reference compound to yield the dynamical instrumental response curve (34).…”

Section: Methodsmentioning

confidence: 99%

Real-time Enzyme Dynamics Illustrated with Fluorescence Spectroscopy of p-Hydroxybenzoate Hydroxylase

Westphal¹,

Matorin²,

Hink³

et al. 2006

Journal of Biological Chemistry

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We have used the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH) to illustrate that a strongly fluorescent donor label can communicate with the flavin via single-pair Förster resonance energy transfer (spFRET). The accessible Cys-116 of PHBH was labeled with two different fluorescent maleimides with full preservation of enzymatic activity. One of these labels shows overlap between its fluorescence spectrum and the absorption spectrum of the FAD prosthetic group in the oxidized state, while the other fluorescent probe does not have this spectral overlap. The spectral overlap strongly diminished when the flavin becomes reduced during catalysis. The donor fluorescence properties can then be used as a sensitive antenna for the flavin redox state. Time-resolved fluorescence experiments on ensembles of labeled PHBH molecules were carried out in the absence and presence of enzymatic turnover. Distinct changes in fluorescence decays of spFRET-active PHBH can be observed when the enzyme is performing catalysis using both substrates p-hydroxybenzoate and NADPH. Single-molecule fluorescence correlation spectroscopy on spFRET-active PHBH showed the presence of a relaxation process (relaxation time of 23 s) that is related to catalysis. In addition, in both labeled PHBH preparations the number of enzyme molecules reversibly increased during enzymatic turnover indicating that the dimer-monomer equilibrium is affected.Single-molecule fluorescence spectroscopy applied to proteins or nucleic acids have allowed direct observations of molecular properties, individual reaction steps, or intermediates that are otherwise hidden in conventional, ensemble experiments (1-5). These techniques have been developed for freely diffusing molecules as well as for surfacebound systems. A powerful approach is single-pair fluorescence resonance energy transfer (spFRET), 2 in which the resonance energy transfer efficiency from the donor to the acceptor of a doubly labeled system is used to distinguish subpopulations with different transfer efficiencies caused by different relative distances and orientations of donor and acceptor molecules arising from conformational changes (see for example Refs. 1, 2, and 6 -8). Single-molecule fluorescence detection techniques have resulted in measurements of single enzyme turnovers of, among others, nuclease (9), horseradish peroxidase (10), ribozyme (11), exonuclease (12), and F 0 F 1 -ATP synthase (13,14).A spectacular breakthrough in single-molecule fluorescence detection of flavoenzymes was reported for cholesterol oxidase, in which single enzymatic turnovers were observed in real-time by monitoring the redox state via FAD (flavin) fluorescence (3, 4). The cholesterol oxidase was confined in an agarose gel preventing the enzyme diffusing away, but allowing free exchange of much smaller sized substrates and products. The flavin cofactor of cholesterol oxidase is nonfluorescent in the reduced state and slightly fluorescent in the oxidized state. Therefore, the successive "on-times" and "off-times" reflect th...

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Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii. A multidimensional time-resolved polarized fluorescence study

Cited by 64 publications

References 38 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

Flavin dynamics in oxidized Clostridium beijerinckii flavodoxin as assessed by time‐resolved polarized fluorescence

Real-time Enzyme Dynamics Illustrated with Fluorescence Spectroscopy of p-Hydroxybenzoate Hydroxylase

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