Femtosecond dynamics of flavoproteins: Charge separation and recombination in riboflavine (vitamin B ₂ )-binding protein and in glucose oxidase enzyme

Abstract

Flavoproteins can function as hydrophobic sites for vitamin B2 (riboflavin) or, in other structures, with cofactors for catalytic reactions such as glucose oxidation. In this contribution, we report direct observation of charge separation and recombination in two flavoproteins: riboflavin-binding protein and glucose oxidase. With femtosecond resolution, we observed the ultrafast electron transfer from tryptophan(s) to riboflavin in the riboflavin-binding protein, with two reaction times: Ϸ100 fs (86% component) and 700 fs (14%). The charge recombination was observed to take place in 8 ps, as probed by the decay of the charge-separated state and the recovery of the ground state. The time scale for charge separation and recombination indicates the local structural tightness for the dynamics to occur that fast and with efficiency of more than 99%. In contrast, in glucose oxidase, electron transfer between flavin-adenine-dinucleotide and tryptophan(s)͞tyrosine(s) takes much longer times, 1.8 ps (75%) and 10 ps (25%); the corresponding charge recombination occurs on two time scales, 30 ps and nanoseconds, and the efficiency is still more than 97%. The contrast in time scales for the two structurally different proteins (of the same family) correlates with the distinction in function: hydrophobic recognition of the vitamin in the former requires a tightly bound structure (ultrafast dynamics), and oxidation-reduction reactions in the latter prefer the formation of a chargeseparated state that lives long enough for chemistry to occur efficiently. Finally, we also studied the influence on the dynamics of protein conformations at different ionic strengths and denaturant concentrations and observed the sharp collapse of the hydrophobic cleft and, in contrast, the gradual change of glucose oxidase. D ynamical processes in proteins play a crucial role in biological structure-function correlations (1, 2), and one such process is electron transfer (ET). In biological systems, ET reactions are ubiquitous (3, 4), especially in enzymes with redox reactions (5). Flavoproteins with flavin chromophores are examples of such enzymes and are involved in various catalytic processes (6-8). The understanding of ET reactions of flavins in proteins and their redox reactions is critical to the enzyme function.In this contribution, we report our femtosecond studies of ET dynamics of riboflavin (RF; vitamin B 2 ) in RF-binding protein (RfBP) and flavin-adenine-dinucleotide (FAD) in glucose oxidase (GOX) ( Fig. 1 and Scheme 1). High-resolution x-ray crystallographic structures of both proteins have recently been reported, and the binding structures of flavins in the proteins are well determined (9-11). RfBP is a globular monomeric protein of approximate dimensions 50 ϫ 40 ϫ 35 Å with a single polypeptide chain of 219 amino acids, nine disulfide bridges, six ␣-helices, and four series of discontinuous areas of ␤ structure.The ligand-binding domain of RfBP is a hydrophobic cleft, Ϸ20 Å wide and 15 Å deep. The binding of RF occurs in the cleft with...

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin

“…The DADS of the time constant 3 shows a significant amplitude at 500 nm and is therefore assigned to the decay of the difference signal at this wavelength. A similar signal is not seen for other flavin-binding proteins (9). A definitive assignment of this difference signal cannot be given, yet.…”

“…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).…”

“…Eukaryotic RfbPs typically bind riboflavin between the aromatic residues of mostly tryptophan-and tyrosine-built triads of stacked aromatic rings. Mechanistically, fast electron transfer from the aromatic moiety to the riboflavin has been proposed to quench the excited state of riboflavin by building a couple of a positively charged amino acid radical and a negatively charged flavin semiquinone (2,9). Ultrafast electron transfer mechanisms from an aromatic moiety to a photoexcited flavin are not only observed for RfbP, but for other flavoproteins, like for BLUF (blue light sensing using FAD) domains, cryptochromes, and DNA photolyases (10 -13).…”

Excited-state dynamics of the fluorescent probe Lucifer Yellow in liquid solutions and in heterogeneous media

“…64 Such conformation has already been observed between riboflavin and Trp in the crystal structure of the riboflavin-riboflavin-binding protein complex. 65 It also reduces the water accessible area. The lack of this water-hiding driving force could explain why no such static fluorescence quenching of LYen by Ind (Trp was not soluble) was detected in DMSO (data not shown).…”

Dissection of complex protein dynamics in human thioredoxin

“…To understand such complex dynamics, we need to dissect the process into elementary steps and determine their relevant time scales. Many elementary reactions even occur on a similar time scale (8) and different methods are often integrated to separate each reaction channel and thus elucidate their molecular mechanisms (5,(9)(10)(11)(12)(13). Here, we report our direct studies of complex protein dynamics at the active site in human thioredoxin (hTrx).…”