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

Abstract

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.

The phrA gene of Rhodobacter sphaeroides encodes a photolyase and is regulated by singlet oxygen and peroxide in a σ E-dependent manner

“…1). Photolyases provide three conserved tryptophan residues, which were supposed to be involved in electron transport to reduce the FADH 2 (Byrdin et al, 2003, Fig. 1).…”

“…PCC6803 (Brudler et al, 2003;bottom line). The black boxed tryptophans are believed to be involved in intraprotein electron transport (Byrdin et al, 2003). The lengths of the proteins are indicated in parentheses.…”

Proton-Coupled Electron Transfer in Organic Synthesis: Fundamentals, Applications, and Opportunities

“…In the former, Brettel has demonstrated that FAD initiates oxidation of Trp382, which engages in a hole-hopping oxidation of Trp359 and finally Trp306, which deprotonates to yield the neutral tryptophan radical [1,[178][179]. For the oxidation of the terminal Trp306 found in E. coli photolyase, Schelvis and coworkers have demonstrated the electron transfer proceeds by concerted PCET below pH = 6.5, but otherwise undergoes stepwise PCET via an initial electron transfer [180].…”

Could tyrosine and tryptophan serve multiple roles in biological redox processes?

“…It is well established that multi-step tunnelling, called hopping, is required for functional charge transport in many redox enzymes (examples include ribonucleotide reductase [1][2][3][4][5][6][7][8][9], photosystem II [10][11][12], DNA photolyase [13][14][15][16][17][18][19][20][21], MauG [22][23][24][25] and cytochrome c peroxidase [26,27]). Here, we advance the hypothesis that many such enzymes, most especially those that generate high-potential intermediates during turnover, could be irreversibly damaged if the intermediates are not inactivated in some way.…”