Delocalized hole transport coupled to sub-ns tryptophanyl deprotonation promotes photoreduction of class II photolyases.

Class II photolyases utilize for the photoreduction of their flavin cofactor (FAD) a completely different tryptophan triad than most other photolyases and cryptochromes. To counter sped-up back electron transfer, they evolved an unusually fast deprotonation of the distal tryptophanyl radical cation (WH˙+) that is produced after excitation of the flavin. We studied the primary aspects of oxidized FAD photoreduction by ultrafast transient absorption spectroscopy, using the class II photolyase from Methanosarcina mazei. With a time constant of 9.2 ps, the initial reduction step of the excited flavin by the proximal W381 tryptophan proceeds almost twentyfold slower than in other photolyases carrying oxidized FAD, most likely because of the larger distance between the flavin and the proximal tryptophan. The thus formed W381H˙+ radical is tracked by transient anisotropy measurements to migrate in 29 ps with delocalization over several members of the tryptophan triad. This 29 ps phase also includes the decay of a small fraction of excited flavin, reacting on a slower timescale, and partial recombination of the FAD˙-/WH˙+ radical pair. A final kinetic phase in 230 ps is assigned to the deprotonation of W388H˙+ that occurs in competition with partial charge recombination. Interestingly, we show by comparison with the Y345F mutant that this last phase additionally involves oxidation of the Y345 phenolic group by W388H˙+, producing a small amount of neutral tyrosyl radical (YO˙). The rate of this electron transfer step is about six orders of magnitude faster than the corresponding oxidation of Y345 by the deprotonated W388˙ radical. Unlike conventional photolyases, where the electron hole accumulates on the distal tryptophan before the much slower tryptophanyl deprotonation, our data show that delocalized hole transport is concomitantly concluded by ultrafast deprotonation of W388H˙+.

Structure of the bifunctional cryptochrome aCRY from Chlamydomonas reinhardtii.

Photolyases and cryptochromes form an almost ubiquitous family of blue light photoreceptors involved in the repair and maintenance of DNA integrity or regulatory control. We found that one cryptochrome from the green alga Chlamydomonas reinhardtii (CraCRY) is capable of both, control of transcript levels and the sexual cycle of the alga in a positive (germination) and negative manner (mating ability), as well as catalyzing the repair of UV-DNA lesions. Its 1.6 Å crystal structure shows besides the FAD chromophore an aromatic tetrad that is indispensable in animal-like type I cryptochromes for light-driven change of their signaling-active redox state and formation of a stable radical pair. Given CraCRY's catalytic activity as (6-4) photolyase in vivo and in vitro, we present the first co-crystal structure of a cryptochrome with duplex DNA comprising a (6-4) pyrimidine-pyrimidone lesion. This 2.9 Å structure reveals a distinct conformation for the catalytic histidine His1, H357, that challenges previous models of a single-photon driven (6-4) photolyase mechanism.

Sub-nanosecond tryptophan radical deprotonation mediated by a protein-bound water cluster in class II DNA photolyases.

Class II DNA photolyases are flavoenzymes occurring in both prokaryotes and eukaryotes including higher plants and animals. Despite considerable structural deviations from the well-studied class I DNA photolyases, they share the main biological function, namely light-driven repair of the most common UV-induced lesions in DNA, the cyclobutane pyrimidine dimers (CPDs). For DNA repair activity, photolyases require the fully reduced flavin adenine dinucleotide cofactor, FADH-, which can be obtained from oxidized or semi-reduced FAD by a process called photoactivation. Using transient absorption spectroscopy, we have examined the initial electron and proton transfer reactions leading to photoactivation of the class II DNA photolyase from Methanosarcina mazei. Upon photoexcitation, FAD is reduced via a distinct (class II-specific) chain of three tryptophans, giving rise to an FAD˙- TrpH˙+ radical pair. The distal Trp388H˙+ deprotonates to Trp388˙ in 350 ps, i.e., by three orders of magnitude faster than TrpH˙+ in aqueous solution or in any previously studied photolyase. We identified a class II-specific cluster of protein-bound water molecules ideally positioned to serve as the primary proton acceptor. The high rate of Trp388H˙+ deprotonation counters futile radical pair recombination and ensures efficient photoactivation.

Nicotinamide Adenine Dinucleotides Arrest Photoreduction of Class II DNA Photolyases in FADH˙ State.

All light-sensitive members of the photolyase/cryptochrome family rely on FAD as catalytic cofactor. Its activity is regulated by photoreduction, a light-triggered electron transfer process from a conserved tryptophan triad to the flavin. The stability of the reduced flavin depends on available external electron donors and oxygen. In this study, we show for the class II photolyase of Methanosarcina mazei, MmCPDII, that it utilizes physiologically relevant redox cofactors NADH and NADPH for the formation of the semiquinoid FAD in a light-dependent reaction. Using redox-inert variants MmCPDII/W388F and MmCPDII/W360F, we demonstrate that photoreduction by NADH and NADPH requires the class II-specific tryptophan cascade of MmCPDII. Finally, we confirmed that mutations in the tryptophan cascade can be introduced without any substantial structural disturbances by analyzing crystal structures of MmCPDII/W388F, MmCPDII/W360F and MmCPDII/Y345F.

Divalent Cations Increase DNA Repair Activities of Bacterial (6-4) Photolyases.

The (6-4) photolyases of the FeS-BCP group can be considered as the most ancient type among the large family of cryptochrome and photolyase flavoproteins. In contrast to other photolyases, they contain an Fe-S cluster of unknown function, a DMRL chromophore, an interdomain loop, which could interact with DNA, and a long C-terminal extension. We compared DNA repair and photoreduction of two members of the FeS-BCP family, Agrobacterium fabrum PhrB and Rhodobacter sphaeroides RsCryB, with a eukaryotic (6-4) photolyase from Ostreococcus, OsCPF, and a member of the class III CPD photolyases, PhrA from A. fabrum. We found that the low DNA repair effectivity of FeS-BCP proteins is largely stimulated by Mg2+ and other divalent cations, whereas no effect of divalent cations was observed in OsCPF and PhrA. The (6-4) repair activity in the presence of Mg2+ is comparable with the repair activities of the other two photolyases. The photoreduction, on the other hand, is negatively affected by Mg2+ in PhrB, but stimulated by Mg2+ in PhrA. A clear relationship of Mg2+ dependency on DNA repair with the evolutionary position conflicts with Mg2+ dependency of photoreduction. We discuss the Mg2+ effect in the context of structural data and DNA binding.

Rhodobacter sphaeroides CryB is a bacterial cryptochrome with (6-4) photolyase activity.

Photolyases are efficient DNA repair enzymes that specifically repair either cyclobutane pyrimidine dimers or (6-4) photoproducts in a light-dependent cleavage reaction. The closely related classical cryptochrome blue light photoreceptors do not repair DNA lesions; instead they are involved in regulatory processes. CryB of Rhodobacter sphaeroides was until now described as a cryptochrome that affects light-dependent and singlet oxygen-dependent gene expression and is unusual in terms of its cofactor composition. Here we present evidence for a repair activity of (6-4) photoproducts by CryB and suggest a dual character combining the functions of cryptochromes and photolyases. We investigated the effects of crucial amino acids involved in cofactor or DNA lesion binding on the light-dependent recovery of cells after UV light exposure (in vivo photoreactivation). Remarkably, impairment of one of the two light absorbing cofactors, FAD or 6,7-dimethyl-8-ribityllumazine, only marginally affected the final survival rate but strongly decelerated photoreactivation kinetics. The impairment of both of them together through mutagenesis decreased CryB-dependent photoreactivation to the level of the ∆cryB knockout strain. The third cofactor, a [4Fe4S] iron-sulfur cluster, is indispensable for the structural integrity of the protein. The reduction of FAD via the conserved tryptophan W338, which is crucial for in vitro reduction and consequently DNA repair, is not required for in vivo photoreactivation, suggesting that this reduction pathway to FAD is dispensable in the cellular environment. This demonstrates that in vitro experiments give only limited information on in vivo photolyase activity.