Femtosecond Fluorescence Dynamics of Flavoproteins:  Comparative Studies on Flavodoxin, Its Site-Directed Mutants, and Riboflavin Binding Protein Regarding Ultrafast Electron Transfer in Protein Nanospaces

Abstract

We have studied the fluorescence dynamics of “nonfluorescent” flavoproteins including flavodoxin (FD), its mutants W60F, Y98F, and W60F/Y98F, and riboflavin binding protein (RBP) with the femtosecond fluorescence up-conversion method and have observed the fluorescence quenching dynamics of FD and its mutants for the first time. The strong fluorescence quenching in these flavoproteins seems to be caused by ultrafast electron transfer (ET) from aromatic amino acid residues to the excited flavin chromophore in stacked configuration according to previous transient absorption studies. In the present work, we have made comparative studies on the dynamics of fluorescence quenching due to ET to the excited chromophore in RBP and FD. We have observed also fluorescence dynamics of FD mutants where active electron donors Trp·NH and Tyr·OH are partially (either of them) or completely replaced by inactive phenylalanine and directly demonstrated the ET mechanism of the ultrafast fluorescence quenching in PNS of FD.

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin

“…Furthermore, the pattern of positive and negative absorbance changes shows distinctive differences: the intensive negative signal of the stimulated emission at 520 -600 nm observed for free riboflavin in aqueous solution is missing for HsDod A -bound riboflavin (2), and the positive signal does not reach that far into the long wavelength region above 700 nm. A discussed mechanism for the fast quenching of the excited state, on the basis of previous investigations on flavoproteins (9,18,19), is an ultrafast electron transfer from a tryptophan residue to the excited flavin. If such an electron transfer occurs, the spectrum should show the absorption characteristics of a cationic tryptophan radical, expected around 560 nm (38,39) and of an anionic flavosemiquinone around 510 nm (40 -42).…”

Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor

“…The picosecond time scale of these events indicates that the residue(s) involved in the transfer must be in close proximity to the isoalloxazine ring of the flavin. Previous studies on flavo-enzymes have revealed that rapid light-driven ET may occur from aromatic residues to flavin (25,26). Tyr-8 (Tyr-21 in AppA) is a good candidate because it is the closest aromatic residue to the flavin and has been shown to be critical for photocycling activity in BLUF domains (12,15,16).…”

Dissection of the triple tryptophan electron transfer chain in Escherichia coli DNA photolyase: Trp382 is the primary donor in photoactivation

“…This result sets it apart from most other flavoproteins, whose flavin cofactor is normally oxidized or fully reduced and whose catalytic mechanism does not require light. ''Artificial'' electron transfer starting from excited oxidized or fully reduced flavins has been studied in several flavoproteins (18)(19)(20), whereas redox photochemistry of flavin radicals is so far only known for photolyase.…”