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Bioelectromagnetism

 

CRY4 has been found in non-mammalian vertebrates, but it lacks the circadian transcriptional regu­

latory function (Kobayashi et al., 2000; Kubo et al., 2006; Takeuchi et al., 2016) or the photorepair activ­

ity (Kobayashi et al., 2000). Tough the function of CRY4 is not well understood, chicken CRY4 (cCRY4)

may be a magnetoreceptor because of its high level of expression in the retina and light-dependent

structural changes in retinal homogenates. To further characterize the photosensitive nature of cCRY4,

Mitsui et al. (2015) developed an expression system using budding yeast and purifed cCRY4 at yields of

submilligrams of protein per liter with binding of the FAD chromophore.

By short durations of cCRY4 irradiation with blue light, Mitsui et al. (2015) detected reduction of the

FADox chromophore to FADH^•, which was not observed in the previous study by Ozturk et al. (2009).

Extended durations of irradiation reduced FADH^• to the FADH⁻ form. Mitsui et al. (2015) detected for the

frst time that the FADH⁻ form returned to FADox in the dark via FADH^• formation, and they could depict

the putative photocycle of cCRY4 (Figure 4.12a). Although the dark oxidation of FADH⁻ to the FADH^•

form was observed in the previous study by Ozturk et al. (2009), their observations are not fully congruent

with the photocycle (Figure 4.12a), probably because there are many diferences in experimental condi­

tions such as the diferent irradiation wavelengths (UV-A or blue light), the light intensity, the expression

system used, the presence of the FLAG tag, and perhaps the concentration of dissolved oxygen.

Te reduction from FADox to FADH is likely composed of two steps: (1) electron transfer to FADox

to generate FAD^•− anion radical and (2) its protonation to generate the neutral FADH^• form (Liu et al.,

2010). Mitsui et al. (2015) did not observe temperature dependency in this reduction process, implying

that both of these steps might be composed of temperature-independent mechanisms under the experi­

mental conditions.

Mitsui et al. (2015) further estimated the absolute absorbance spectra of FADox, FADH, and FADH^•−

forms (Figure 4.12b) using the diference absorption spectra obtained from our present measurements.

Ten, by using the photon fuence rate, the absorbance of the sample, and the rate of photoreaction of

FADox to FADH^• (blue light irradiation), Mitsui et al. (2015) could roughly estimate the quantum yield

for the photoreduction (Φ1) to be ≈3%. Similarly, the quantum yield for the photoreduction of FADH^•

to FADH⁻ (Φ2) (red light irradiation) was estimated to be ≈2%. Tese values are lower than the quan­

tum yields for the photoreduction of the other CRYs such as Chlamydomonas aCRY (animal-like CRY)

(Φ1 ≈ 7%) (Spexard et al., 2014), but these values may be enough to receive an external light or magnetic

signals accounting for the wide and relatively strong expression of cCRY4 in the retina.

Te speculated photocycle of cCRY4 (Figure 4.12a) is similar to that of AtCRY1 (Lin et al., 1995), but in

the case of AtCRY1, FADH^• was not detected spectroscopically when FADH⁻ was incubated in the dark

for reoxidation to FADox (Müller and Ahmad, 2011). Tis may be due to the rapid oxidation of FADH^•

to FADox in the two-electron reoxidation process of AtCRY1. In this study, both the FADH^• and FADH⁻

forms of cCRY4 were oxidized in vitro under dark conditions. A recent study of chicken CRY1a (cCRY1a)

(Nießner et al., 2013), another CRY identifed in the chicken retina, implied that cCRY1a may absorb not

only blue light but also that of longer wavelengths (e.g., green and yellow) utilizing the FADH^• state and

changing its CRY C-terminal extension (CCE) conformation in the FADH⁻ state. Nießner et al. (2013)

analyzed cCRY1a activation using the chicken retina in vivo; therefore, future investigation extending

our novel yeast expression system to other CRY proteins both in vivo and in vitro may be benefcial to

further analyses.

Concerning the biological functions of CRY4, avian CRYs are thought to work as light-driven mag­

netoreceptors (Ritz et al., 2000) based on their localization in the retina (Mouritsen et al., 2001; Nießner

et al., 2011; Watari et al., 2012), and the photosensitivity of the purifed protein (Liedvogel et al., 2007;

Du et al., 2014). On the other hand, in the Western clawed frog (Xenopus tropicalis), CRY4 is highly

expressed in the ovary and testis rather than the retina (Takeuchi et al., 2016), and hence more likely

to be implicated in unknown photic function(s) in the gonadal tissues instead of magnetoreception.

Considering that CRY involves multiple functions such as nonvisual photoreception (Emery et al., 1998;

Tu et al., 2004), magnetoreception (Gegear et al., 2008), and vision (Mazzotta et al., 2013), CRY4 may play

multiple roles in diferent cells and/or organs.