Determination of rates and yields of interchromophore (folate .fwdarw. flavin) energy transfer and intermolecular (flavin .fwdarw. DNA) electron transfer in Escherichia coli photolyase by time-resolved fluorescence and absorption spectroscopy

111

116

DNA photolyase is a pyrimidine-dimer repair enzyme that uses visible light. Photolyase generally contains two chromophore cofactors. One is a catalytic cofactor directly contributing to the repair of a pyrimidine-dimer. The other is a light-harvesting cofactor, which absorbs visible light and transfers energy to the catalytic cofactor. Photolyases are classified according to their second cofactor into either a folate- or deazaflavin-type. The native structures of both types of photolyases have already been determined, but the mechanism of substrate recognition remains largely unclear because of the lack of structural information regarding the photolyase-substrate complex. Photolyase from Thermus thermophilus , the first thermostable class I photolyase found, is favorable for function analysis, but even the type of the second cofactor has not been identified. Here, we report the crystal structures of T. thermophilus photolyase in both forms of the native enzyme and the complex along with a part of its substrate, thymine. A structural comparison with other photolyases suggests that T. thermophilus photolyase has structural features allowing for thermostability and that its light-harvesting cofactor binding site bears a close resemblance to a deazaflavin-type photolyase. One thymine base is found at the hole, a putative substrate-binding site near the catalytic cofactor in the complex form. This structural data for the photolyase-thymine complex allow us to propose a detailed model for the pyrimidine-dimer recognition mechanism.

mentioning

confidence: 99%

mentioning

confidence: 99%

Crystal structure of thermostable DNA photolyase: Pyrimidine-dimer recognition mechanism

Kakemoto

Masui

Kuramitsu

et al. 2001

111

116

“…Photolyase binds specifically to damaged DNA. After complex formation, the repair reaction is presumably initiated by electron transfer from photoexcited FADH Ϫ to the pyrimidine dimer (17).…”

mentioning

confidence: 99%

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

Byrdin

Eker

Vos

et al. 2003

100

150

In Escherichia coli photolyase, excitation of the FAD cofactor in its semireduced radical state (FADH • ) induces an electron transfer over Ϸ15 Å from tryptophan W306 to the flavin. It has been suggested that two additional tryptophans are involved in an electron transfer chain FADH • 4 W382 4 W359 4 W306. To test this hypothesis, we have mutated W382 into redox inert phenylalanine. Ultrafast transient absorption studies showed that, in WT photolyase, excited FADH • decayed with a time constant Ϸ 26 ps to fully reduced flavin and a tryptophan cation radical. In W382F mutant photolyase, the excited flavin was much longer lived ( Ϸ 80 ps), and no significant amount of product was detected. We conclude that, in WT photolyase, excited FADH • is quenched by electron transfer from W382. On a millisecond scale, a product state with extremely low yield (Ϸ0.5% of WT) was detected in W382F mutant photolyase. Its spectral and kinetic features were similar to the fully reduced flavin͞neutral tryptophan radical state in WT photolyase. We suggest that, in W382F mutant photolyase, excited FADH • is reduced by W359 at a rate that competes only poorly with the intrinsic decay of excited FADH • ( Ϸ 80 ps), explaining the low product yield. Subsequently, the W359 cation radical is reduced by W306. The rate constants of electron transfer from W382 to excited FADH • in WT and from W359 to excited FADH • in W382F mutant photolyase were estimated and related to the donor-acceptor distances.

“…Ultrafast spectroscopic studies have recently been reported in several flavin enzymes (12)(13)(14)(15)(16). The fluorescence quenching of both RfBP and GOX was observed to occur on femtosecond and picosecond time scales by measurements of the excited-state flavin (RF* or FAD*) fluorescence decay (16).…”

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confidence: 99%

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

Zhong

Zewail

2001

218

222

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...