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

Abstract

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.

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

“…4 and 5 ), we decided to examine the behaviour of its W388F mutant, in which the terminal Trp of the triad was replaced by a non-reducing phenylalanine. Our structural data for this and the Y345F mutant (PDB codes ; , ; ) 32 show that there are no compensatory structural changes of residues lining the ET pathway, which could complicate the following analysis and interpretation. The transient absorption signals obtained for the W388F mutant on the ns/μs timescale upon excitation by 5 ns pulses at 470 nm ( Fig.…”

Ultrafast Oxidation of a Tyrosine by Proton-Coupled Electron Transfer Promotes Light Activation of an Animal-like Cryptochrome

“…3,7,25 In the canonical PCSf members, the excited flavin abstracts an electron from a nearby (proximal) tryptophan, which gets in turn reduced by a second (medial) tryptophan, before being reduced by a third (distal) one. The distal tryptophan is usually surface-exposed and can hence be reduced by external electron donors such as thiol compounds 26 or NAD(P)H. 27,28 For animal-like cryptochromes and photolyases, a fourth aromatic residue, Y 373 in CraCRY and W 360 in the photolyase from Xenopus laevis, has been found to act as final electron donor of an extended electron transfer chain. 24,[29][30][31][32] Transient absorption spectroscopy (TAS) has been extensively used to study the mechanistic details of the photoreduction of photolyases 31,[33][34][35][36][37][38][39][40] and cryptochromes, taking advantage of the rather distinct absorption spectra of the species involved ( Figure 1B).…”

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

“…In particular, CPD photolyases are subdivided into three classes: class I photolyases are the most well-known and occur in microbes, class II photolyases are predominantly found in higher, multicellular eukaryotes and class III photolyases occur in some eubacteria. Class II photolyases are particularly interesting as they exhibit significant deviations in terms of sequence and structure of the photocatalytic site, 7,[11][12][13][14] as well as of their photorepair mechanism. 15 All photolyases non-covalently bind a chromophoric cofactor, flavin adenine dinucleotide (FAD), which is essential for the DNA repair process and adopts a unique U-shaped conformation.…”

“…Notably, the Y345F mutation was shown by X-ray crystallography to cause no substantial disturbance of the crystal structure. 14 Furthermore, distinguishing between putative WH •+ intermediates as expected to be transiently formed within the tryptophan triad during the charge migration process is not straightforward because they have the same chemical nature and hence similar spectroscopic properties. As in several previous studies, 19,22,25,27,28,34 polarized transient absorption spectroscopy was used here to access the orientations of the involved species through the associated transient anisotropy.…”