Extended Electron-Transfer in Animal Cryptochromes Mediated by a Tetrad of Aromatic Amino Acids

Abstract

The cryptochrome/photolyase protein family possesses a conserved triad of tryptophans that may act as a molecular wire to transport electrons from the protein surface to the FAD cofactor for activation and/or signaling-state formation. Members from the animal (and animal-like) cryptochrome subclade use this process in a light-induced fashion in a number of exciting responses, such as the (re-)setting of circadian rhythms or magnetoreception; however, electron-transfer pathways have not been explored in detail yet. Therefore, we present an in-depth time-resolved optical and electron-paramagnetic resonance spectroscopic study of two cryptochromes from Chlamydomonas reinhardtii and Drosophila melanogaster. The results do not only reveal the existence of a fourth, more distant aromatic amino acid that serves as a terminal electron donor in both proteins, but also show that a tyrosine is able to fulfill this very role in Chlamydomonas reinhardtii cryptochrome. Additionally, exchange of the respective fourth aromatic amino acid to redox-inactive phenylalanines still leads to light-induced radical pair formation; however, the lifetimes of these species are drastically reduced from the ms- to the μs-range. The results presented in this study open up a new chapter, to our knowledge, in the diversity of electron-transfer pathways in cryptochromes. Moreover, they could explain unique functions of animal cryptochromes, in particular their potential roles in magnetoreception because magnetic-field effects of light-induced radical pairs strongly depend on distance and orientation parameters.

On the optimal relative orientation of radicals in the cryptochrome magnetic compass

“…The hyperfine mechanism applies here despite the immobilisation of the radicals in the protein matrix, because an efficient charge-separation pathway exists that yields a well-separated radical pair in which inter-radical interactions are weak [18] compared to the dominating hyperfine interactions. In the mentioned in vitro experiments, this pair consists of a flavin adenine dinucleotide co-factor (FAD, here abbreviated as F) anion and a cation derived from the third tryptophan (W) of a highly-conserved electron transfer chain (the so-called tryptophan triad/tetrad [19]; see Figure 1A), i.e. [F •− W •+ ].…”

“…These arise from static inhomogeneity, which is expected to manifest within the ensemble of contributing sensory proteins in a realistic biological environment [40]. Tyrosine radicals (abbreviated as Y • ) have also been implicated within cryptochrome photo-chemistry [19,[48][49][50]. However, no detailed theoretical studies of the magnetosensitivity of [F •− Y • ] have so far been presented.…”

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

“…In some cases, this tryptophan triad serving as electron transfer chain is extended by an additional tryptophan 27,29 or tyrosine residue. [30][31][32] When starting with the semi-reduced flavin, the onset of photoreduction is significantly slower, in about 30 ps, and may become the rate limiting step of the overall charge separation process. 4 Deprotonation of the distal WH •+ radical to the neutral W • species then follows on a much longer timescale, e.g.…”

Ascorbic acid may not be involved in cryptochrome-based magnetoreception

“…DmCry and ErCry1a were investigated in three redox states: (i) the dark state (DS) that exists prior to the photo-induced charge separation, ( [3,9,10,21,22] (Fig. 1).…”

“…Animal cryptochromes incorporate a chain of three or four tryptophan residues [3,9,10], shown in Fig. 1 and conventionally labelled, in order of increasing distance from the FAD, as WA, WB, WC and WD.…”