J. Phys. Chem. B 2010, 114, 10717–10727
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A Fluorescence Perspective on the Differential Interaction of Riboflavin and Flavin Adenine Dinucleotide with Cucurbit[7]uril Sharmistha Dutta Choudhury,* Jyotirmayee Mohanty, Achikanath C. Bhasikuttan, and Haridas Pal* Radiation & Photochemistry DiVision, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India ReceiVed: May 7, 2010; ReVised Manuscript ReceiVed: July 2, 2010
The interaction of the macrocyclic host, cucurbit[7]uril (CB7), with riboflavin (RF) and its derivative, flavin adenine dinucleotide (FAD), has been investigated using absorption and steady-state and time-resolved fluorescence measurements. Interestingly, in the presence of CB7, the fluorescence intensity of RF is quenched, whereas the fluorescence intensity of FAD is enhanced. It is proposed that the fluorescence quenching of RF results from the tautomerization of its isoalloxazine moiety from the lactam to the lactim forms, upon binding to CB7. Such a tautomerization can be brought about since the two lactim forms have higher dipole moments than the lactam form of RF, and thus experience much stronger dipole-dipole interactions (and hence greater binding affinities) with CB7 in the former cases than in the latter. This tautomerization in the presence of CB7 leads to a significant reduction in the observed radiative decay rate and hence a reduction in the fluorescence intensity of RF. Binding of CB7 with RF is confirmed by an increase in the rotational correlation time of RF in the presence of CB7. Geometry optimization studies indicate the formation of an exclusion complex between CB7 and RF, possibly stabilized by H-bonding interactions, as also suggested by the characteristic red shift in the absorption spectra of the CB7-RF system. In the case of FAD, both the isoalloxazine ring and the adenine moiety can interact with the CB7 host. In aqueous solutions, a good fraction of FAD molecules exists in a “closed” conformation with the adenine and isoalloxazine rings stacked together, thus leading to very efficient fluorescence quenching due to the ultrafast intramolecular electron transfer from adenine to the isoalloxazine moiety. Binding of the adenine and/or the isoalloxazine moiety of FAD with CB7 inhibits the stacking interaction and changes the “closed” conformation to the “open” conformation, wherein the adenine and isoalloxazine moieties are widely separated, thus prohibiting the electron transfer process. This reduces the inherent fluorescence quenching of FAD molecule and results in the observed fluorescence enhancement. As observed for RF, the interaction of CB7 with the isoalloxazine ring of FAD should cause fluorescence quenching due to the lactam to lactim tautomerization process. However, in the interplay between the above two opposing effects, the fluorescence enhancement due to the modulation in the conformational dynamics of FAD by the CB7 host predominates. The conformational change is in fact supported by the observation of a long lifetime component in the fluorescence decay of FAD in the presence of CB7. Moreover, at acidic pH, when FAD is already present mainly in the “open” form, the conformational dynamics no longer plays any major role and the fluorescence of FAD is quenched by CB7, as expected, due to the tautomerization at the isoalloxazine moiety. 1. Introduction Supramolecular host-guest interaction is rapidly emerging as the method of choice for tuning molecular properties to meet desired applications like drug delivery vehicles, sensors, designing molecular architectures, catalysis chambers, biomimetic systems, and so on.1-8 Presently, the repertoire of host systems available is quite vast, the most commonly studied macrocyclic hosts being the different homologues and derivatives of cyclodextrins, cucurbiturils, and calixarenes.9-11 Different types of investigations with these systems have led to a good understanding about the mode of their interactions with a variety of guest molecules, the primary driving forces responsible for the complexation, and about the prospective applications of such host-guest interactions.9-15 Several groups, including ours, have demonstrated many of the aforementioned interesting phenomena through host-guest interactions like changes in acid-base * Corresponding authors. E-mail:
[email protected] (S.D.C.);
[email protected] (H.P.). Fax: 91-22-25505151/25519613.
behavior of encapsulated guests,5,16 guest relocation from host cavity to biomolecular binding pocket,17 supramolecular architecture formation,2,6,18 biological catalysis,3 sensors,19,20 and fluorescent capsule formation.21 In a previous communication, we reported on the modulation of the intramolecular electron transfer process in flavin adenine dinucleotide, upon interaction with the β-cyclodextrin (β-CD) host.22 Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which consist of a heterocyclic isoalloxazine moiety tethered to a ribityl phosphate or ribityl adenine diphosphate chain, respectively, are the most commonly occurring flavins in flavoproteins (Scheme 1a). These flavin cofactors are derivatives of riboflavin (RF), a compound better known as vitamin B2 (Scheme 1b).23 Because of their chemical versatility, flavoproteins are ubiquitous and participate in a broad spectrum of biological activities.24,25 Flavoproteins are the ideal systems for studies of intraprotein electron transfer and conformational dynamics of biomacromolecules, not only because the flavin (isoalloxazine) moiety is a redox-active group suitably located
10.1021/jp1041662 2010 American Chemical Society Published on Web 08/04/2010
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J. Phys. Chem. B, Vol. 114, No. 33, 2010
Dutta Choudhury et al.
SCHEME 1: Chemical Structures of (a) FAD, (b) RF (Different Tautomeric Forms), and (c) CB7a
a
Major dimensions of interest for these systems are also indicated.
in the heart of the active site but also due to the fact that it is a fluorescent chromophoric group, which makes it amenable for various fluorescence studies.26-29 The fluorescence spectral characteristics as well as the fluorescence quantum yield of flavins strongly depend on environmental factors like solvent polarity and refractive index.30-32 Although RF and FMN have reasonably high fluorescence quantum yields (Φf ) 0.26) in aqueous solutions, FAD is very weakly fluorescent (Φf ) 0.03).33 The remarkably low fluorescence yield of FAD compared to RF or FMN was first reported by Weber, and it is now well understood that the reduction in the fluorescence quantum yield of FAD results from both static and dynamic quenching of the flavin fluorescence due to the photoinduced electron transfer from the adenine moiety to the isoalloxazine moiety.34-40 On the basis of different studies, like circular dichroism,41 NMR,42 ultraviolet resonance Raman spectroscopy,43 and MD simulations,40 it is confirmed that, in solution, FAD exists in two conformations: an extended or “open” conformation in
which the isoalloxazine and the adenine moieties are largely separated from each other and a “closed” conformation in which the two aromatic rings are in close proximity and stacked together.34-40 The “closed” conformation is preferred in aqueous solutions, and is stabilized by the combined effect of the π-π interaction between the isoalloxazine ring and the adenine moiety and the intramolecular H-bonding interactions along the phosphate sugar backbone.35 Sequence-structure relationships of several FAD binding proteins have revealed that, in most of these proteins, the FAD cofactor is bound in an extended manner except in some members of the ferredoxin reductase family and in the DNA photolyase enzyme, where the cofactor adopts a bent conformation.44 Although there is no explicit correlation between the conformation of FAD and the activity of the flavoproteins, there have been some indications of a functional implication of the FAD conformation.45,46 Quantum chemical calculations on the FAD cofactor in DNA photolyase, which is an enzyme that catalyzes photorepair of UV damaged DNA by
Differential Interaction of RF and FAD with CB7 an electron transfer mechanism, suggest that, due to the bent conformation of FAD, the electron transfer between flavin and the thymine bases takes place indirectly with the adenine moiety acting as an intermediate.46 Femtosecond time-resolved studies, on the other hand, rule out any direct involvement of the adenine group in the electron transfer repair mechanism of DNA photolyase but suggest that the adenine moiety mediates the repair by anchoring the thymine dimer through H-bonds and thus modulates the electron jump by a superexchange mechanism.47 Very recently, Acocella et al. have used a quantum mechanical time-dependent approach to show that the adenine moiety in photolyase provides the necessary electrostatic interactions to promote the electron transfer and sterically keeps the influence of the surrounding medium under control.48 Thus, in our previous study, we felt it was quite intriguing to investigate the conformational changes of FAD as well as the modulation in its photophysical properties and intramolecular electron transfer behavior in the presence of the biomimetic binding pockets of CD hosts. Our results revealed that the binding of β-CD to FAD leads to a change in its conformation from the “closed” to the “open” form.22 This conformational change increases the distance between the electron donor (adenine) and the electron acceptor (isoalloxazine) moieties, and thus inhibits the electron transfer process, which in turn leads to a significant fluorescence enhancement as well as an increase in the fluorescence lifetime of FAD. Comparative studies with the parent molecule, RF, showed no characteristic changes, thus indicating that the β-CD host binds preferably to the adenine moiety of FAD rather than to its isoalloxazine ring. In the present work, we have investigated the interaction of RF and FAD with another versatile macrocyclic host, cucurbit[7]uril (CB7, Scheme 1c), using absorption, steady-state fluorescence, and time-resolved fluorescence measurements. Although the dimensions of the host cavities are quite similar for both β-CD and CB7, they have large structural and functional dissimilarities, which lead to the diverse behavior of the two macrocyclic hosts. While CB7 is a cyclic polymer of seven glycoluril units, β-CD is composed of seven glucopyranose units. Unlike β-CD, which is shaped like a torus and whose ends are laced with hydroxyl groups, CB7 is a symmetrical pumpkin shaped molecule having carbonyl laced portals. This variation in the composition of the peripheral functional groups leads to significant differences in the nature of interactions of β-CD and CB7 hosts with various guest molecules. Quite understandably, due to the presence of the carbonyl groups at the portals of CB7, the dipole-dipole and/or ion-dipole interactions play a major role in the stable complex formation of CB7 with suitable guest molecules. In the present study, interactions between the CB7 host and the biologically important molecules, RF and FAD, have been investigated, considering the present hostguest systems as an alternative and simple model for the enzymesubstrate interactions in biological systems. 2. Experimental Section Riboflavin (RF) and cucurbit[7]uril (CB7) were obtained from Aldrich and used as such. FAD was obtained from Sigma and used after purification by ion exchange chromatography for the removal of the possible degradation products (FMN and RF) in the sample. Thus, the FAD sample from Sigma (purity 96%) was eluted at an ion strength gradient between 1 and 10 mM phosphate buffer (pH 7.0) using a DEAE Sephacil column (Sigma). The concentrations of RF and FAD were calculated from their molar extinction coefficients (ε450, RF ) 12200 M-1cm-1, ε450, FAD ) 11300 M-1cm-1)49 and were maintained
J. Phys. Chem. B, Vol. 114, No. 33, 2010 10719 in the range 4-10 µM in all of the experimental solutions. The interaction of RF or FAD with CB7 was studied by adding different weighed amounts of CB7 to the respective fluorophore solutions. All studies with FAD at pH 7 were performed in 10 mM phosphate buffer in Nanopure water (Millipore Elix3/A10 water purification system; conductivity of KA, it follows that K2 > K1 and K2′ > K1′ (see Note S1 in the Supporting Information). In analyzing the fluorescence titration curve, however, it is quite justified to consider an effective binding constant K for all of the tautomeric forms, as given by eq 2 earlier, which can be more explicitly written as eq 8. K
RFlactam + CB7 y\z CB7 • RFlactam/lactims
(8)
It should be stressed here that the complexation of RF with CB7 is not likely to convert the lactam form completely to the lactim forms; rather, a good fraction of the complexes should be present in the CB7•RFlactam form. It is important to mention further that the value of the equilibrium constant, K ∼6700 M-1, as estimated from the analysis of the fluorescence titration curve, can at the most be considered as the effective binding constant for all of the different tautomeric forms of RF (Note S1, Supporting Information). Considering that KB, KB′ > KA, the observed binding constant is expected to be greater than KA, which is the true binding constant of the lactam form with CB7. We feel that the lactam to lactim conversion of RF in the presence of CB7, which arises due to the higher binding affinity of CB7 with the lactim forms, is the reason for the reduction in the fluorescence intensity of RF upon interaction with CB7. However, the question that arises is why the lactim forms should have a better binding affinity for the CB7 host than the lactam form. It is known that the dipole-dipole interaction plays a major role in the binding of various guest molecules with the CB7 host. Thus, if the dipole moments of the lactim forms are larger than that of the lactam form, a preferential interaction of the lactim forms with CB7 can be anticipated. To have qualitative support for this idea, we have theoretically estimated the ground state dipole moments for the different tautomeric forms of RF by PM3 calculations with molecular mechanics (MM) correction using the Gaussian 92 package.52 These values are listed in Table 2. It is seen that both of the lactim forms have higher dipole moments than that of the lactam form. Geometry optimization studies were also carried out to have
some insight on the mode of interaction of RF with CB7 and to have an estimate of the relative stabilities of the host-guest complexes. The ∆Hf values for the complex formation as estimated from the optimized parameters are listed in Table 2, and the optimized geometries are presented in Figure 4. The ∆Hf values clearly show that the CB7 complexes with the lactim forms, which have higher dipole moments, are more stable than the CB7 complex with the lactam form of RF. These results thus support our proposition for the CB7 induced lactam to lactim tautomerization of RF. The geometry optimized structures further indicate that, in the present systems, exclusion type complexes (rather than inclusion complexes) are formed for both the lactam (A) and lactim (B and B′) forms of RF with CB7. It is proposed that the exclusion complexes are possibly stabilized by the H-bonding interactions of the carbonyl portals of CB7, with the -NH group in the lactam form and the -OH groups in the lactim forms of RF. Such H-bonding interaction is in fact supported by the observed red shift in the 370 nm absorption band in the presence of the CB7 host. It has been suggested in the literature that in biological systems the isoalloxazine moieties are capable of undergoing tautomeric conversion into the enolic forms as a result of the H-bonding interactions with proteins.63,64 An analogous situation thus seemed to prevail in the presence of the macrocyclic host, CB7. 3.2. Absorption, Steady-State Fluorescence, and TimeResolved Fluorescence Changes of FAD in the Presence of CB7. The absorption spectra of FAD in the absence and presence of CB7 are presented in Figure 5 after correction for the background absorption by the respective CB7 concentrations. Similar to RF, two absorption bands are observed with maxima around 450 and 370 nm, corresponding to the S0 to S1 and S0 to S2 transitions of the isoalloxazine chromophore, respectively. The changes in the absorption spectra with increasing CB7 concentrations are qualitatively similar to those of RF. A red shift of the 370 nm band is observed. However, unlike RF, no clear isosbestic point is observed in the present case due to a larger decrease in absorbance for both of the bands. The changes in the absorption spectra indicate that the isoalloxazine ring of FAD interacts with the CB7 host. Like RF, the fluorescence spectrum of FAD is broad with maximum emission around 530 nm (Figure 6). However, in this case with increasing CB7 concentration, there is an enhancement in the fluorescence intensity, unlike the fluorescence quenching observed in the case of RF (cf. Figure 2). In addition to the isoalloxazine ring, the FAD molecule has another potential site for interaction with CB7, namely, the adenine moiety. The dimension of the adenine moiety (∼4.8 Å, cf. Scheme 1) is also quite compatible for a possible inclusion within the CB7 cavity. In fact, in our earlier study on the interaction of FAD with the β-CD host, we observed that the major host-guest interaction takes place via the inclusion of the adenine moiety of FAD into the β-CD cavity. Since the size of the host cavity for CB7 is quite similar to that of β-CD, in the CB7-FAD system also, a sizable interaction is expected via the inclusion of the adenine moiety into the CB7 cavity. This interaction, however, is not likely to cause any effect on the absorption and fluorescence spectra of FAD, since the basic chromophore unit in FAD is its isoalloxazine ring. Considering the interaction of CB7 with isoalloxazine, it is expected that the fluorescence intensity should reduce rather than increase, as observed in the case of RF, due to the proposed lactam-lactim tautomerization induced by CB7. Thus, the observed enhancement in the fluorescence intensity for the CB7-FAD system suggests that, in addition to the above tautomerization process,
Differential Interaction of RF and FAD with CB7
J. Phys. Chem. B, Vol. 114, No. 33, 2010 10723
Figure 4. Geometry optimized structures for the complexes of CB7 with the different tautomeric forms of RF: (a) CB7•RFlactam; (b) CB7•RFlactim,B; (c) CB7•RFlactim,B′.
Figure 5. Representative absorption spectra of FAD (5 µM) in 10 mM phosphate buffer at pH 7 with different CB7 concentrations, after correction for the background absorption by CB7. [CB7]/mM: (1) 0.0, (2) 0.10, (3) 0.33, (4) 0.45, (5) 0.79, (6) 1.0.
Figure 6. Steady-state fluorescence spectra of FAD (5 µM) in 10 mM phosphate buffer (pH 7) at different CB7 concentrations. [CB7]/mM: (1) 0.0, (2) 0.20, (3) 0.45, (4) 0.76, (5) 1.0, (6) 1.5. The excitation wavelength was 420 nm. The inset shows the increase in the fluorescence intensity of FAD with respect to CB7 concentration. As explained in the text, no reasonable binding isotherm could be constructed for any satisfactory quantitative analysis.
there must be some other mechanism which leads to a significant increase in the fluorescence intensity of FAD on its interaction with CB7. As mentioned earlier, an enhancement in the fluorescence intensity of FAD was observed in our previous study on its interaction with β-CD, in spite of a very weak binding in the host-guest system. In this case, the fluorescence enhancement was explained in terms of the conformational change of FAD from the “closed” to the “open” form on its binding (via the inclusion of the adenine moiety) with the β-CD host. A similar situation is also very likely for the CB7-FAD system, although in the present case FAD possibly interacts with CB7 involving both the adenine and the isoalloxazine moieties. It is reported
that, in aqueous solution, a large fraction of the FAD molecules in the ground state exists in the “closed” conformation, where the isoalloxazine ring and the adenine moiety are stacked on each other and this stacking-unstacking process is dynamic in nature and occurs in the time scale of about 19 ns.38,65 The proximity of the isoalloxazine and the adenine moieties in the “closed” conformer results in the efficient fluorescence quenching due to an ultrafast photoinduced intramolecular electron transfer from the adenine to the isoalloxazine moiety.35,37,38,40,65 In the presence of CB7, the steric repulsion between the CB7 complexed adenine and/or isoalloxazine moieties will lead to a large change in the conformational dynamics of FAD, causing the stacking-unstacking equilibrium to shift largely toward the “open” conformation. This conformational change will thus significantly reduce the photoinduced electron transfer from the adenine moiety to the isoalloxazine ring due to an increase in the distance between the electron donor and acceptor groups, and accordingly will reduce the inherent fluorescence quenching observed in the free FAD molecule. Recent studies by Radoszkowicz et al. based on fluorescence measurements and MD simulations have shown that the fluorescence quenching due to intramolecular electron transfer is possible only when the distance between the adenine and the isoalloxazine moieties is
