Inhibiting Intramolecular Electron Transfer in Flavin Adenine Dinucleotide by Host-Guest Interaction: A Fluorescence Study Noufal Kandoth, Sharmistha Dutta Choudhury,* Jyotirmayee Mohanty, Achikanath C. Bhasikuttan, and Haridas Pal* Radiation & Photochemistry DiVision, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India ReceiVed: October 14, 2009; ReVised Manuscript ReceiVed: December 24, 2009 Modulation in the photophysical properties and intramolecular electron transfer behavior of the flavin adenine dinucleotide (FAD) molecule has been investigated in the presence of the macrocyclic hosts, R-, - and γ-cyclodextrins (CDs), using absorption and steady-state and time-resolved fluorescence measurements. The results demonstrate that only the -CD host has a suitable cavity dimension to form a weak inclusion complex with FAD by encapsulating the adenine moiety, which is the preferred binding site in the large FAD molecule. Interestingly, in spite of the weak binding interaction, a significant enhancement in the fluorescence intensity of FAD is observed on complexation with -CD, and this has been attributed mainly to the modulation in the conformational dynamics of FAD in the presence of -CD. In aqueous solutions, a good fraction of FAD molecules exist in a “closed” conformation with the adenine and isoalloxazine rings stacked on each other, thus leading to very efficient fluorescence quenching due to the ultrafast intramolecular electron transfer from adenine to the isoalloxazine moiety. Complex formation with -CD inhibits this intramolecular electron transfer by changing the “closed” conformation of FAD to the “open” form, wherein the adenine and isoalloxazine moieties are widely separated, thus prohibiting the fluorescence quenching process. Further evidence for the conformational changes has been obtained by the observation of a long lifetime component in the fluorescence decay of FAD in the presence of -CD, which corresponds to the decay of the unquenched “open” form of FAD. Fluorescence up-conversion studies also indicate the absence of any ultrafast component in the fluorescence decay arising from the complexed FAD, thus supporting the formation of the “open” form in the presence of -CD, with no intramolecular electron transfer. 1. Introduction The photochemistry and photophysics of flavins have drawn considerable research interest due to their essential role in many light-driven biological activities. 1–16 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). 2 Flavin can have three different redox states: oxidized form, one- electron reduced radical semiquinone, and two-electron fully reduced hydroquinone. 3 Because of their chemical versatility, flavoproteins are ubiquitous and participate in a broad spectrum of biological activities. 16,17 Flavoproteins are the ideal systems for studies of intraprotein electron transfer and conformational dynamics of biomacromolecules, not only because the flavin moiety is a redox-active group suitably located in the heart of the active site, but also because it has a fluorescent chromophoric group, thus making it amenable for fluorescence studies. 6,15,18,19 The fluorescence spectral characteristics as well as the fluorescence quantum yield of flavins strongly depend on the environmental factors such as refractive index and solvent polarity. 1,12,20 Although RF and FMN have reasonably high fluorescence quantum yields (Q ) 0.26) in aqueous solutions, FAD is very weakly fluorescent (Q ) 0.03). 11 The remarkably low fluorescence yield of FAD compared to RF or FMN was first reported by Weber and was proposed to be due to the formation of an intramolecular ground-state complex between the isoalloxazine ring and the adenine moiety of FAD. 11 It is now well understood that the reduction in the quantum yield of FAD results from both static and dynamic quenching of the flavin fluorescence due to photoinduced electron transfer from the adenine moiety to the isoalloxazine moiety. 4,9,13,14,21–23 On enzymatic digestion of the diphosphate bridge of the FAD molecule, the fluorescence intensity increases substantially to match with that of the free FMN. Based on these studies, it was proposed that, in solution, FAD exists in two conformations: an extended or “open” form 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. 4,9,13,14,21–23 The “closed” conformation is sta- bilized by the combined effect of the π-π interaction between the isoalloxazine ring and the adenine moiety and the intramo- lecular hydrogen bonding interactions along the phosphate sugar backbone. 4 Support for the presence of stacked conformation has been obtained from circular dichroism, 24 NMR studies, 25 ultraviolet resonance Raman spectroscopy 26 and molecular dynamics (MD) simulations, 13 leading to different structural models for the interactions between the flavin and adenine moieties. 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 it adopts a bent conformation. 27 * Corresponding author. E-mail: sharmidc@barc.gov.in (S.D.C.); hpal@barc.gov.in (H.P.). Fax: 91-22-25505151/25519613. J. Phys. Chem. B 2010, 114, 2617–2626 2617 10.1021/jp909842z  2010 American Chemical Society Published on Web 02/04/2010