Abstract
Flavin adenine dinucleotide (FAD) is an important cofactor in many light-sensitive enzymes. The role of the adenine moiety of FAD in light-induced electron transfer was obscured, because it involves an adenine radical, which is short-lived with a weak chromophore. However, an intramolecular electron transfer from adenine to flavin was revealed several years ago by RobertKaptein by using chemically induced dynamic nuclear polarization (CIDNP). The question of whether one or two types of biradicals of FAD in aqueous solution are formed stays unresolved so far. In the present work, we revisited the CIDNP study of FAD using a robust mechanical sample shuttling setup covering a wide magnetic field range with sample illumination by a light-emitting diode. Also, a cost efficient fast field cycling apparatus with high spectral resolution detection up to 16.4 T for nuclear magnetic relaxation dispersion studies was built based on a 700 MHz NMR spectrometer. Site-specific proton relaxation dispersion data for FAD show a strong restriction of the relative motion of its isoalloxazine and adenine rings with coincident correlation times for adenine, flavin, and their ribityl phosphate linker. This finding is consistent with the assumption that the molecular structure of FAD is rigid and compact. The structure with close proximity of the isoalloxazine and purine moieties is favorable for reversible light-induced intramolecular electron transfer from adenine to triplet excited flavin with formation of a transient spin-correlated triplet biradical F-A. Spin-selective recombination of the biradical leads to the formation of CIDNP with a common emissive maximum at 4.0 mT detected for adenine and flavin protons. Careful correction of the CIDNP data for relaxation losses during sample shuttling shows that only a single maximum of CIDNP is formed in the magnetic field range from 0.1 mT to 9 T; thus, only one type of FAD biradical is detectable. Modeling of the CIDNP field dependence provides good agreement with the experimental data for a normal distance distribution between the two radical centers around 0.89 nm and an effective electron exchange interaction of 2.0 mT.
Highlights
Flavins play an important role as coenzymes in various biological systems and have been studied extensively
Nuclear magnetic relaxation dispersion (NMRD) experiments were run with 4.4 mM Flavin adenine dinucleotide (FAD) solution in D2O, pH 3.9, using a 700 MHz Bruker Avance III HD NMR spectrometer equipped with a TXI probe and a home-made fast field cycling add-on, similar to the one which has been built earlier (Zhukov et al, 2018)
The closed conformation was obtained in our density-functional theory (DFT) calculations of the 3D structure of a triplet excited FAD molecule in aqueous solution using the Gaussian program package (Frisch et al, 2009)
Summary
Flavins play an important role as coenzymes in various biological systems and have been studied extensively. Flavin adenine dinucleotide (FAD) attracted much attention in the last decades as a cofactor of the cryptochrome photoreceptor that is suggested to be responsible for sensitivity to the Earth’s magnetic field in animal and avian navigation (Wiltschko and Wiltschko, 2019). A review on the radical-pair mechanism (RPM) of magnetoreception as a leading hypothesis to explain bird navigation can be found in the literature (Hore and Mouritsen, 2016). The keystone of the proposed explanation is as follows: the blue-light-activated flavin moiety of FAD oxidizes a chain of three tryptophan compounds resulting in a radical pair composed of a singly reduced semiquinone flavin and an oxidized tryptophan. The singlet/triplet spin dynamics of the FAD−/Trp+ radical pair have been intensively studied as the source of cryptochrome sensitivity to the Earth’s magnetic field
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