Bioelectromagnetism History, Foundations and Applications (Shoogo Ueno, Tsukasa Shigemitsu)

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Bioelectromagnetism

non-covalently to the chromophore, FAD and possibly the second chromophore, 5,10-methenyltetrahy-

drofolate (MTHF) (Lin et al., 1995; Malhotra et al., 1995; Banerjee et al., 2007; Klar et al., 2007).

For some animal CRYs, it can even be the primary photoproduct after blue light irradiation (Song

et al., 2006; Liu et al., 2010). Here, FAD^•⁻ (FAD* in Figure 4.9) is a semi-reduced anion radical species

in which electron transfer alone occurs. When FAD is irradiated with blue light, tryptophan (Trp) is

able to act as an electron donor due to relatively higher occupied molecular orbital (HOMO) energy

level. In addition to Trp, tyrosine (Tyr) is an amino acid that causes photoexcited electron transfer with

respect to FAD. The highest HOMO energy level of these amino acids are higher than the HOMO of

FAD. Therefore, when FAD is photoexcited, electron transfer occurs from the amino acid HOMO to the

vacant orbit of FAD, and a charge-separated state and a radical pair are formed.

Recent studies have shown that these differences depend not only on Trp but also on what amino

acids are located near the FAD (Giovani et al., 2003; Liedvogel et al., 2007; Berndt et al., 2007; Damiani

et al., 2010; Iwata et al., 2010; Kattnig and Hore, 2017; Hammad et al., 2020). The structure has been

investigated in the known CRY, especially for amino acids that affect the protonation of FAD. It has been

reported that mutating asparagine (Asn), near the FAD of Synechocystis CRY-DASH (ScyCRY-DASH)

(Brudler et al., 2003), to cysteine (Cys) stabilizes the flavin radical state FAD^•⁻ (Iwata et al., 2010). Here,

CRY-DASHs (Drosophila, Arabidopsis, Synechocystis, human-type cryptochromes) form one subclade

of the cryptochrome/photolyase family (CPF), and CPF members are flavoproteins that act as DNA-

repair enzymes (DNA-photolyases), or as UV-A/blue-light photoreceptors (cryptochromes) (Kiontke

et al., 2020). The name CRY-DASH was invented by Brudler et al. (2003), together with the discovery of

another photolyase homolog in Arabidopsis thaliana (AtCRY3; AT5G24850.1) that contained FAD and

did bind to undamaged dsDNA and single-stranded (ss) DNA, but was unable to complement photo-

reactivation in photolyase-deficient E. coli cells (Kleine et al., 2003). It has also been reported that the

original natural protein stabilizes the flavin radical state FADH^• more than the mutant protein in which

Asn, in the vicinity of the FAD of photolyase (flavoprotein having a function of repairing DNA), was

replaced by Cys (Damiani et al., 2010).

In the case of the amino acid sequences of natural CRY and photolyase in the Drosophila melano-

gaster CRY (DmCRY), Cys is located in the vicinity of FAD (Iwata et al., 2010). In this case, the flavin

radical state FAD^•⁻ is stabilized and has the function of a circadian clock (Iwata et al., 2010). The form of

FAD in the circadian clock for Drosophila melanogaster is regarded as FAD^•− (Kattnig and Hore, 2017,

Figure 4.11).

The FAD cofactor leads to the formation of radical pairs via sequential electron transfers along the

“tryptophan (Trp)-triad,” a chain of three conserved Trp residues within the protein (Zeugner, et al.,

2005; Biskup et al., 2009; Giovani et al., 2003). This process reduces the photoexcited singlet state of the

FAD to the anion radical, FAD^•⁻, and oxidizes the terminal, surface-exposed, Trp (TrpCH) to give the

cation radical, TrpCH^•+ (Kattnig and Hore, 2017). Formed with conservation of spin angular momentum,

FIGURE 4.11 Electron transfer pathway in cryptochromes in the Drosophila melanogaster (DmCRY) (Kattnig

and Hore, 2017). After photoexcitation of the FAD cofactor, three or four rapid sequential electron transfers along

a triad or tetrad of tryptophan (Trp) residues (WA, WB, WC, WD) generate a spin-correlated radical pair [FAD⁻

TrpCH⁺] or [FAD⁻ TrpDH⁺]. The figure is based on the crystal structure of DmCRY (PDB ID: 4GU5) (Zoltowski et al.,