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...

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.

We have recently characterized the role of the riboflavinbinding protein (RfBP) dodecin from Halobacterium salinarum as clearing free riboflavin from the cytoplasm with riboflavin protein dissociation constants in the low nanomolar range, [1] and as providing riboflavin as the direct precursor for FMN and FAD biosynthesis. To prevent cellular damage, dodecin seals riboflavin in deeply buried binding cavities and neutralizes riboflavin reactivity by extensively quenching photoactivated states. [2] Both properties are achieved by a remarkable binding mode. Binding to dodecin, riboflavin aligns into a sandwich of aromatic systems in which extensive stacking compensates for minimal hydrogen bonding ( Figure 1). In the key step of the relaxation process of the light-activated riboflavin, an electron of tryptophan W36 is transferred to the excited flavin, generating a charge-separated intermediate state that subsequently recombines to the ground state. Recently, we were able to assign time constants to the individual processes in the photocycle of dodecin: 1) charge separation faster than the resolution of the experiment (< 0.2 ps, t 1 ); 2) electron back-transfer with a time constant of 0.9 ps (t 2 ); (3) a relaxation process with 6 ps parallel to (2) with an intermediate absorbing at 500 nm (t 3 ); and (4) proton transfer from the surrounding water coupled with the electron-transfer/back-transfer cycle (Scheme 1). [3] Based on high-resolution X-ray structural data and a concise functional characterization, establishing a system of extraordinarily well-defined structure-function relationships, we considered dodecin as excellently suited for modulating biological electron-transfer reactions by rational protein design, thereby studying the protein in a manipulative manner.This approach should be achieved by exchanging the native W36 with analogues of varying ionization potential leading to W36-riboflavin pairs of modulated redox potential difference. Given the computed value of 7.42 eV for the indole unit of tryptophan at the DFT/B3LYP/6-31G* level of theory in the gas phase, [4] we chose the derivatives 4-aminotryptophan (4NH 2 -W), 4-fluorotrypthophan (4F-W), and 4azatryptophan (4Aza-W) for shifting the ionization potential to 6.68, 7.49, and 7.96 eV, respectively (see Supporting Information). The C-4 position of the flavin-holding W36 was chosen for derivatization, as X-ray structural analysis indicated an empty bulge in the binding pocket allowing noninvasive modification. To prepare noncanonical W36-modified dodecin analogues, we applied the supplementationbased incorporation method (SPI) [5] together with the established procedures for dodecin purification and folding Figure 1. Sandwich-like incorporation of flavins. In dodecin, dimers of riboflavin (Rf) are bound between tryptophans (W36) and aligned antiparallelly by glutamines (Q55) at the interface of two protomers related by C 2 symmetry.

Employing nonequilibrium molecular dynamics (MD) simulations and transient infrared (IR) spectroscopy, a joint theoretical/experimental study on a water-soluble photoswitchable octapeptide designed by Renner et al. [Biopolymers 2002, 63, 382] is presented. The simulations predict the cooling of the hot photoproducts on a time scale of 7 ps and complex conformational rearrangements ranging from a few picoseconds to several nanoseconds. The experiments yield a dominant fast relaxation time of 5 ps, which is identified as the cooling time of the peptide in water and also accounts for initial conformational changes of the system. Moreover, a weaker component of 300 ps is found, which reflects the overall conformational relaxation of the system. The virtues and the limitations of the joint MD/IR approach to describe biomolecular conformational rearrangements are discussed.

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Gezähmte Elektronen: Es galt, einen Photozyklus gezielt zu verändern. Dazu wurde das Flavoprotein Dodecin in der Schlüsselaminosäure Tryptophan in endoskopischer Weise mit Substituenten – gewählt nach strukturellen und elektronischen Einflüssen – verändert. Dieser Ansatz ist ideal in der Präzision des rationalen Protein‐Engineerings und erlaubt die Korrelation des Ionisierungspotentials von Tryptophan und Elektrontransferraten in einem Marcus‐Modell.

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