Photoactivation of the flavin cofactor in Xenopus laevis (6–4) photolyase: Observation of a transient tyrosyl radical by time-resolved electron paramagnetic resonance

Abstract

The light-induced electron transfer reaction of flavin cofactor photoactivation in Xenopus laevis (6 -4) photolyase has been studied by continuous-wave and time-resolved electron paramagnetic resonance spectroscopy. When the photoactivation is initiated from the fully oxidized form of the flavin, a neutral flavin radical is observed as a long-lived paramagnetic intermediate of two consecutive single-electron reductions under participation of redox-active amino acid residues. By time-resolved electron paramagnetic resonance, a spin-polarized transient radical-pair signal was detected that shows remarkable differences to the signals observed in the related cyclobutane pyrimidine dimer photolyase enzyme. In (6 -4) photolyase, a neutral tyrosine radical has been identified as the final electron donor, on the basis of the characteristic line width, hyperfine splitting pattern, and resonance magnetic field position of the tyrosine resonances of the transient radical pair. U ltraviolet light (Յ300 nm) damages cellular DNA by formation of cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts from adjacent pyrimidine bases on the same DNA strand (1). Such dimers may be restored to their monomeric form through the action of two photoactive (300ϽϽ500 nm) damage-specific DNA repair enzymes, named CPD photolyase (also called DNA photolyase) (2-6) and (6-4) photolyase (7,8). Both enzymes are found in various organisms and exhibit a 20-30% amino acid sequence identity (3, 9, 10). Two photoactive pigments are used in the DNA repair pathway. One is invariably a redox-active FAD (11,12), and the other so-called second chromophore, which acts as a light harvester, is a methenyltetrahydrofolate in most species (11,13,14) but 5-deazariboflavin in certain rare species that synthesize this compound (15,16). It has been proposed that the initial step in the DNA repair mechanism is a photoinduced single electron transfer (ET) from the FAD cofactor, which in the active enzyme is in its fully reduced form (12,17,18), FADH Ϫ , to the DNA lesion. This mechanism is supported by a previous electron paramagnetic resonance (EPR) study in which a photoinduced spin-polarized radical pair (RP) signal assigned to the flavin-CPD dimer complex was observed (19).If the enzyme is found in an inactive state with FAD either semireduced as neutral radical, FADH ⅐ , or fully oxidized, FAD ox (20), photolyases can undergo reversible ET reactions with the participation of amino acid residues to lower the redox state of the flavin cofactor to FADH Ϫ . This photoactivation process has recently attracted much experimental (20-26) and theoretical (27, 28) interest. In Escherichia coli CPD photolyase, cofactor photoactivation proceeds on a nanosecond time scale via a chain of tryptophan residues (W-382, -359, and -306) (29). W-306 is believed to be the final electron donor that is rereduced on a millisecond time scale either by back ET from the flavin or by exogenous reductants (30). In Anacystis nidulans CPD photolyase, however, a further ET step from a t...

Blue Light Perception in Plants

“…Hence, the competing electron transfer reaction observed in the mutant must be much less efficient and, hence, slower to ensure that only FMN-cysteinyl adduct formation occurs. This is in contrast to other flavoenzymes such as the structurally unrelated photolyases, where the photoreduction from the fully oxidized flavin proceeds on a nanosecond time scale (28). It would be surprising, therefore, if the crucial step in adduct formation were, as previously postulated (20), a proton transfer rather than an electron transfer, given that it should be significantly faster than the alternative electron transfer process observed in the C450A mutant.…”

“…This is even more evident when taking into account the small size of the LOV2 domain (23). Electron transfer time constants in the sub-microsecond range are observed in other flavoproteins; for example, in photolyases in which electrons are transferred over distances exceeding the diameter of LOV domains (28). This demonstrates that, in contrast to many other flavoproteins, low activation energy electron transfer pathways do not exist in the LOV2 domain or that endergonic steps in the electron transfer pathway have to be considered.…”

Magnetically sensitive light-induced reactions in cryptochrome are consistent with its proposed role as a magnetoreceptor

“…The latter is not seen here probably because it is obscured by the recombination of RP1 which appears to be faster in AtCry than in the algal protein. Formation of tyrosyl radicals has been reported in AtCry (9, 10) and in two photolyases (25,27), but, at least for AtCry, on a much slower (approximately 1 ms) timescale (9) than the changes in Fig. 2A which, if due to a tyrosyl radical, would have to be assigned to its reduction by an unknown electron donor following its formation at an unprecedented fast rate.…”

Tyrosine Radical Formation in the Reaction of Wild Type and Mutant Cytochrome P450cam with Peroxy Acids

“…47) and in aerobic RNR (36,48). They were also found to be transient intermediates in a variety of enzymes, such as peroxidases (31), cytochrome c oxidase (49), and in DNA photolyase, where, depending on the organism, tryptophan or tyrosine radicals have been observed (50). The reported g x values of tyrosine radicals range from 2.0091 for a non-polar environment in RNR of E. coli (34, 38, 51) to 2.0076 for a hydrogenbonded situation in RNR of mouse and yeast (34,38,39) and in plant photosystem II (37,43).…”