Evidence for Concerted Electron Proton Transfer in Charge Recombination between FADH^– and ³⁰⁶Trp^• in Escherichia coli Photolyase

Abstract

Proton-coupled electron-transfer (PCET) is a mechanism of great importance in protein electron transfer and enzyme catalysis, and the involvement of aromatic amino acids in this process is of much interest. The DNA repair enzyme photolyase provides a natural system that allows for the study of PCET using a neutral radical tryptophan (Trp(•)). In Escherichia coli photolyase, photoreduction of the flavin adenine dinucleotide (FAD) cofactor in its neutral radical semiquinone form (FADH(•)) results in the formation of FADH(-) and (306)Trp(•). Charge recombination between these two intermediates requires the uptake of a proton by (306)Trp(•). The rate constant of charge recombination has been measured as a function of temperature in the pH range from 5.5 to 10.0, and the data are analyzed with both classical Marcus and semi-classical Hopfield electron transfer theory. The reorganization energy associated with the charge recombination process shows a pH dependence ranging from 2.3 eV at pH ≤ 7 and 1.2 eV at pH(D) 10.0. These findings indicate that at least two mechanisms are involved in the charge recombination reaction. Global analysis of the data supports the hypothesis that PCET during charge recombination can follow two different mechanisms with an apparent switch around pH 6.5. At lower pH, concerted electron proton transfer (CEPT) is the favorable mechanism with a reorganization energy of 2.1-2.3 eV. At higher pH, a sequential mechanism becomes dominant with rate-limiting electron-transfer followed by proton uptake which has a reorganization energy of 1.0-1.3 eV. The observed 'inverse' deuterium isotope effect at pH < 8 can be explained by a solvent isotope effect that affects the free energy change of the reaction and masks the normal, mass-related kinetic isotope effect that is expected for a CEPT mechanism. To the best of our knowledge, this is the first time that a switch in PCET mechanism has been observed in a protein.

Spectroscopic characterization of radicals and radical pairs in fruit fly cryptochrome – protonated and nonprotonated flavin radical‐states

“…When forming FADH À , protonation has to take place to compensate for the second negative charge. Although this is beyond the scope of the present study, altered pK a values of the fully reduced FAD form should promote protonation, perhaps even in a concerted reaction [57]. Our results indicate that the amino acid proximal to N(5) does not shift the flavin redox potential significantly.…”

“…This can be explained by the two identical rate constants (Table ) and a fast unresolved protonation reaction following the initial rate‐limiting electron‐transfer reaction that inhibits an appropriate disentanglement of the two states by global analysis. Rate‐limiting electron transfer followed by fast proton transfer has been observed previously in photoreduction of FADH • in Ec PL E109A . Although the reaction FAD •− → FADH • combined with protonation of the terminal Trp residue was investigated, the rate‐limiting electron transfer in question also occurs along the Trp triad.…”

Photochemistry of Wild‐Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy

“…Lowering the pH from physiological 7.5 to 6.5 had no significant effect on the signals in the 30 μs time window (see Figure S3 in the Supporting Information). Given the remarkable stability of WT Ec PL between pH 5.4 and 9.5 and the striking instability of the N378D mutant protein at pH>8, we would not exclude that degradation/precipitation of N378D mutant Ec PL and its unbinding of FAD at pH>8 may be connected to the deprotonation of D378, which, in that case, would indicate that D378 had a p K a somewhere between 8 and 9 and was hence predominantly protonated in our transient absorption experiments (at pH 7.5 and 6.5). These points may be addressed by molecular dynamics simulations similar to those performed on At CRY1 …”

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

“…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]. Moreover, charge recombination between FADH• and Trp306 is pH dependent, slowing down at increased pH [181-182], presumably in accord with this mechanistic switch.…”