Dodecin Sequesters FAD in Closed Conformation from the Aqueous Solution

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Background: Dodecin prevents riboflavin from autodegradation and exerting photo-induced cellular stress. Results: Ultrafast depopulation of excited states and ground state recovery is observed by transient spectroscopy. Conclusion: Ultrafast electron transfer in combination with proton transfer is responsible for deactivation of flavin excited states. Significance: A comprehensive study of parameters in the binding pocket affecting the riboflavin quenching mechanism is given.

Section: Resultsmentioning

confidence: 99%

“…Structures revealed a dodecameric fold, providing six identical binding pockets, which each enables binding of two antiparallel arranged flavins between two tryptophan residues building an aromatic tetrade arrangement ( Fig. 1A) (1)(2)(3)(4)(5)(6)(7).…”

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confidence: 99%

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin

Staudt

Oesterhelt

Grininger

et al. 2012

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“…Structural studies and fluorescence-based binding experiments for the H. salinarum dodecin (9) showed the binding of several kinds of flavins and flavin-like molecules. Furthermore, lumiflavin and lumichrome bind like FMN and riboflavin as stacked dimers in the flavin binding pockets with 1:1 stoichiometries, whereas FAD is bound in a U-shaped locked conformation with an apododecin/ligand ratio of 2:1 (10). Binding constants obtained by fluorescence titrations indicate preferred binding of small flavins and flavin-like molecules such as lumiflavin and lumichrome compared with the bulkier flavins FMN and FAD.…”

mentioning

confidence: 95%

The Dodecin from Thermus thermophilus, a Bifunctional Cofactor Storage Protein

Meissner

Schleicher

Weber

et al. 2007

Dodecins are so far the smallest known flavoproteins (68 -71 amino acids) and are most likely involved in prokaryotic flavin storage. The dodecin monomers adopt a simple ␤␣␤␤-fold and assemble to hollow sphere-like dodecameric complexes. Flavin binding by the dodecin from Thermus thermophilus showed a 1:1 stoichiometry and apparent dissociation constants in the submicromolar to nanomolar range as characterized by isothermal titration calorimetry and fluorescence titrations. The x-ray structures of the flavin-prebound and FMN-reconstituted state of the T. thermophilus dodecin revealed binding of FMN dimers in a novel si-si-rather than the re-re-orientation of their isoalloxazine moieties as found before in an archaeal dodecin. Electron paramagnetic resonance studies demonstrated that upon reduction the excess electron is localized only on one flavin, thus making dodecin-bound flavins highly refractory to redox chemistry. Besides FMN dimers, trimers of coenzyme A are additionally bound to this eubacterial dodecin along the 3-fold symmetry face II of the dodecin complex. Therefore, dodecins can act as bifunctional cofactor storage proteins that sequester catalytic cofactors in prokaryotes very efficiently in an aggregated and unreactive state.Flavoproteins are ubiquitous proteins using flavins as prosthetic groups. Because riboflavin (vitamin B 2 ) serves mostly as a biosynthetic intermediate, FMN and FAD are the principal flavin cofactors (1). They are involved in the catalysis of a wide range of biological redox reactions, such as the dehydrogenation of NAD(P)H, lipid esters, and D-amino acids, the oxidation of amines to imines, the formation and cleavage of disulfide bonds, the hydroxylation of aromatic substrates, or the activation of molecular oxygen. Furthermore, flavoproteins serve as electron transmitters in electron transfer processes like oxidative phosphorylation. Here, they act as mediators between typical 2-electron donors like NADH and 1-electron acceptors like the heme group. This versatility to engage in one-and twoelectron transfer reactions is due to their isoalloxazine moiety that adopts either an oxidized, a semiquinoid, or fully reduced redox state (2). Flavins are not only involved in redox reactions but also in the sensing of blue or ultraviolet light (3). In cryptochromes, flavin chromophores mediate the flowering and daily light/dark cycles in plants (4), in phototropins they regulate phototropism (5), and in photolyases they are involved in DNA repair (6). Beside catalytic or sensoric actions, flavoproteins are also involved in the binding or transport of flavins. During pregnancy, a specific carrier system evolves in vertebrates including the riboflavin-binding proteins (RCPs or RfBPs), since adequate amounts of vitamin B 2 are essential for the normal fetal development (7).Dodecins are a novel family of flavin-binding proteins of unknown function that were discovered in the Archaea Halobacterium salinarum during an inverse structural genomics project on halophilic proteins (8). So far it...

“…Additional affinity for lumichrome (LCR) could moreover characterize dodecin as a protein with a dual binding mode (12)(13)(14). Based on sequence conservation, dodecins cluster in an archaeal and a bacterial subgroup.…”

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confidence: 99%

Dodecin Is the Key Player in Flavin Homeostasis of Archaea

Grininger

Staudt

Johansson

et al. 2009

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Flavins are employed to transform physical input into biological output signals. In this function, flavins catalyze a variety of light-induced reactions and redox processes. However, nature also provides flavoproteins with the ability to uncouple the mediation of signals. Such proteins are the riboflavin-binding proteins (RfBPs) with their function to store riboflavin for fast delivery of FMN and FAD. Here we present in vitro and in vivo data showing that the recently discovered archaeal dodecin is an RfBP, and we reveal that riboflavin storage is not restricted to eukaryotes. However, the function of the prokaryotic RfBP dodecin seems to be adapted to the requirement of a monocellular organism. While in eukaryotes RfBPs are involved in trafficking riboflavin, and dodecin is responsible for the flavin homeostasis of the cell. Although only 68 amino acids in length, dodecin is of high functional versatility in neutralizing riboflavin to protect the cellular environment from uncontrolled flavin reactivity. Besides the predominant ultrafast quenching of excited states, dodecin prevents light-induced riboflavin reactivity by the selective degradation of riboflavin to lumichrome. Coordinated with the high affinity for lumichrome, the directed degradation reaction is neutral to the cellular environment and provides an alternative pathway for suppressing uncontrolled riboflavin reactivity. Intriguingly, the different structural and functional properties of a homologous bacterial dodecin suggest that dodecin has different roles in different kingdoms of life.Flavins are a major class of cofactors that are able to accept and donate electrons as well as to absorb visible light. Flavins consist of a nonconserved aliphatic moiety, covalently linked to a conserved aromatic isoalloxazine ring ( In all flavins, the catalytically active unit is the isoalloxazine substructure (2-4). To handle the reactivity of the isoalloxazine ring, FMN and FAD are tightly bound to flavoenzymes. Finely tuned binding restricts the reaction spectrum of FMN and FAD to discrete, beneficial reaction pathways, preventing harmful side reactions. Reported functions of flavoenzymes include transferring electrons from and to reactive centers (e.g. respiratory chain) as well as employing light for the induction of radical reactions (e.g. DNA-photolyase) or conformational changes (e.g. phototropin) (5-8). In the biosynthesis, the full catalytic power of flavins has already formed at the stage of the FMN and FAD precursor RF (see Fig. 1). As RF is not used as an enzymatically active compound, it is, different to FMN and FAD, not integrated into flavoenzymes. To avoid a negative impact of uncontrolled flavin reactivity by free RF, nature established sequestering devices termed riboflavin-binding proteins (RfBPs). These proteins have been shown to store and distribute RF in eukaryotes, ensuring a fast supply of FMN and FAD (9, 10).The small flavoprotein dodecin from the archaeon Halobacterium salinarum was reported to bind RF with high affinity and to extensiv...