Directed Manipulation of a Flavoprotein Photocycle

摘要

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, 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. 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, τ1) ; 2) electron back-transfer with a time constant of 0.9 ps (τ2) ; (3) a relaxation process with 6 ps parallel to (2) with an intermediate absorbing at 500 nm (τ3) ; and (4) proton transfer from the surrounding water coupled with the electron-transfer/back-transfer cycle (Scheme 1). 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.