Directed Manipulation of a Flavoprotein Photocycle

Abstract

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.

Spectroscopic evidence for direct flavin-flavin contact in a bifurcating electron transfer flavoprotein

“…Absorbance extends to wavelengths longer than 500 nm (66,67) as in the Tyr/flavin/Trp stack of flavodoxin (71,78) and riboflavin-binding protein (79). In an elegant study wherein Trp analogs varying with respect to electron donating ability were installed in dodecin in place of the stacked Trp, analogs with lower tendency to donate electron density showed less long-wavelength absorbance whereas presence of the better electron donor 4-NH2-Trp resulted in a resolved CT band centred near 670 nm, and accelerated electron transfer to photoexcited flavin (55).…”

“…Dodecins are believed to prevent photodamage catalyzed by flavins by passivating the flavin's intrinsic photoreactivity (80). The binding mode allows retention of the OX flavin's absorbance spectrum but essentially abolishes fluorescence and triplet formation due to very rapid electron transfer from the stacked Trp that outcompetes other events (55,62,67). Similarly rapid electron transfer between two flavins is expected if one were present as the HQ, as we propose here.…”

“…This argues against efficient hole hopping via residue 37. If the side chain of Tyr37.S participates in long-range CT, the band might also be expected to occur at a different wavelength in the Y37W variant (for example, see (55)). Instead, Y37W.S RpaETF yielded a signal at 726 ±2 nm.…”

Molecular Mechanism of Flavin Photoprotection by Archaeal Dodecin: Photoinduced Electron Transfer and Mg2+-Promoted Proton Transfer

“…Intriguingly, HsDod is capable of quenching the photoexcited state of flavins in an ultrafast manner . Differences in structure and function between bacterial and archaeal dodecin have been extensively discussed, whereby HsDod is best understood with respect to its unique structure and function. − ,, …”

“…The excited state quenching mechanism has been experimentally studied by a number of spectroscopic methods. , A photoinduced electron transfer from the adjacent tryptophan residue (W36, Figure b) to the flavin species has been suggested as initial step in the deactivation mechanism . This photoinduced charge separation is well-known in flavin photochemistry. − Furthermore, subsequent proton transfer from surrounding water molecules has been discussed as possible second deactivation channel forming a flavosemiquinone radical.…”