論文の公開元へA deazariboflavin chromophore kinetically stabilizes reduced FAD state in a bifunctional cryptochrome
概要
Title
A deazariboflavin chromophore kinetically
stabilizes reduced FAD state in a bifunctional
cryptochrome
Author(s)
Hosokawa, Yuhei; Morita, Hiroyoshi; Nakamura,
Mai et al.
Citation
Scientific Reports. 2023, 13(1), p. 16682
Version Type VoR
URL
rights
https://hdl.handle.net/11094/93237
This article is licensed under a Creative
Commons Attribution 4.0 International License.
Note
Osaka University Knowledge Archive : OUKA
https://ir.library.osaka-u.ac.jp/
Osaka University
www.nature.com/scientificreports
OPEN
A deazariboflavin chromophore
kinetically stabilizes reduced
FAD state in a bifunctional
cryptochrome
Yuhei Hosokawa , Hiroyoshi Morita , Mai Nakamura & Junpei Yamamoto *
An animal-like cryptochrome derived from Chlamydomonas reinhardtii (CraCRY) is a bifunctional
flavoenzyme harboring flavin adenine dinucleotide (FAD) as a photoreceptive/catalytic center and
functions both in the regulation of gene transcription and the repair of UV-induced DNA lesions in
a light-dependent manner, using different FAD redox states. To address how CraCRY stabilizes the
physiologically relevant redox state of FAD, we investigated the thermodynamic and kinetic stability
of the two-electron reduced anionic FAD state (FADH−) in CraCRY and related (6–4) photolyases. The
thermodynamic stability of FADH− remained almost the same compared to that of all tested proteins.
However, the kinetic stability of FADH− varied remarkably depending on the local structure of the
secondary pocket, where an auxiliary chromophore, 8-hydroxy-7,8-didemethyl-5-deazariboflavin
(8-HDF), can be accommodated. The observed effect of 8-HDF uptake on the enhancement of the
kinetic stability of FADH− suggests an essential role of 8-HDF in the bifunctionality of CraCRY.
While sunlight is indispensable for the evolution of life on Earth, solar energy in the UV-B and -C ranges
(100–315 nm) exerts a harmful effect on DNA by altering the chemical structures of adjacent pyrimidine bases
into lesions represented by cyclobutene pyrimidine dimers (CPDs) and/or pyrimidine(6–4)pyrimidone photoproducts ((6–4)PPs), which potentially cause mutagenesis and c arcinogenesis1,2. To maintain genetic integrity,
many organisms possess photolyases (PLs), flavoproteins able to repair the DNA lesions to their original structures using blue light3. Despite classification of PLs into CPD PLs and (6–4) PLs based on their substrates, bond
rearrangement via photoinduced electron transfer from a flavin adenine dinucleotide (FAD) cofactor to the
substrates is a common key reaction mediated by PLs4,5. While FAD in PLs can be observed in three redox states,
namely, oxidized ( FADox), one-electron reduced neutral ( FADH·), or two-electron reduced anionic ( FADH−)
forms, only the F
ADH− state can be used for the repair reaction. Because of the prerequisite of the F
ADH− state
for their activity, PLs developed a unique process in which FAD is reduced in a light-dependent manner, called
photoreduction6. In addition to the FAD cofactor, an auxiliary chromophore can be found in some PLs and
is known to absorb photon energy, transfer it to FAD through the Förster resonance energy transfer (FRET)
mechanism7, and assist with the photoexcitation of FADH− for the photorepair r eaction8. So far, 8-hydroxy7,8-didemethyl-5-deazariboflavin (8-HDF) has been identified as an antenna chromophore in some (6–4) PLs9–12.
Cryptochromes (CRYs) are phylogenetically relevant to PLs with similar structural architectures but are
involved in various biological events apart from DNA repair, such as circadian rhythm r egulation13. Therefore,
PLs and CRYs form a flavoprotein family called the Photolyase/Cryptochrome Superfamily (PCSf). Several
types of CRYs from plants and animals have been identified as photoreceptive CRYs bearing FAD. For example,
plant CRYs are representative photoreceptors that transmit light signals to the blue light signaling pathways,
which regulate photomorphogenic b
ehaviors14–16 and circadian rhythm entrainment17. In addition to plant CRYs
that have evolved from CPD PLs, Drosophila-type CRYs (dCRYs), which are evolutionarily derived from (6–4)
PLs, have also been characterized as photoreceptors that primarily function in synchronization of the circadian
rhythm with sunlight18. Unlike PLs functioning in the FADH− state, these photoreceptive CRYs appear to interact with other partner proteins in not FADH− but the one-electron reduced form (FADH· for plant CRYs19 or a
one-electron reduced anionic state ( FAD·−) for dCRYs20). Hence, understanding the mechanism by which PCSf
proteins with similar frameworks differently stabilize the FAD redox states required for their biological actions
provides a fundamental clue for the origin of their functional diversity.
Graduate School of Engineering Science, Osaka University, 1‑3 Machikaneyama, Toyonaka, Osaka 560‑8531, Japan.
*
email: yamamoto.junpei.es@osaka-u.ac.jp
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In previous s tudies21–25, the residue next to the N5 atom of isoalloxazine in FAD was found to be related to
the functional redox states of FAD in PCSf proteins. An Asp residue in plant CRYs enables rapid generation of
its active F
ADH· state by protonating F
AD·−22. In contrast, Cys residues in dCRYs reportedly inhibit protonation, yielding an active FAD·− state23. In both CPD and (6–4) PLs, an Asn residue is exclusively conserved at the
position6. In particular, the Asn residue in CPD PLs stabilizes the F
ADH· state in both kinetic and thermodynamic
manner for the repair reaction performed with the F
ADH·/FADH– redox p
air24. A time-resolved crystallographic
study of FAD photoreduction in a PCSf protein captured the transient motion of the Asn residue to stabilize
the FADH· state, thereby acting as a redox sensor triad together with the proximal Arg–Asp salt bridge25. These
examples indicate that the N5-proximal Asp, Asn, and Cys residues exclusively govern the functional FAD state
in PCSf proteins. However, the recent discovery of animal-like CRY from Chlamydomonas reinhardtii (CraCRY)
challenges the paradigm. CraCRY is found to exhibit not only photoreceptive CRY functions regulating the
transcription of genes26 but also the ability to repair (6–4)PPs using FADH−27. Interestingly, CraCRY has an Asn
residue proximal to the N5 atom of isoalloxazine of FAD and adopts F
ADox, FADH·, and FADH− states28 as well
as (6–4) PLs do. Therefore, it is challenging to determine how CraCRY sharing a similar architecture and redox
chemistry of FAD with those of (6–4) PLs executes additional CRY functions.
In this study, we shed light on the molecular origin of the unique functionality of CraCRY by determining
the thermodynamic stability of the FADH− state and its kinetic stability against the reoxidation reaction in
CraCRY and its evolutionarily related (6–4) PLs. Consistent with the conservation of residues around FAD, the
thermodynamic stability did not show a significant difference between them. However, the kinetic stability of
FADH− in the tested proteins varied remarkably depending on the local structure of the secondary pocket, where
natural antenna chromophores, such as 8-HDF, can be accommodated. Based on these results, we propose that
the bifunctionality of CraCRY is likely regulated by the presence or absence of 8-HDF.
Results
Evaluation of thermodynamic stability of F
ADH− in (6–4) PLs
We first evaluated the thermodynamic stability of F
ADH− in (6–4) PLs from Arabidopsis thaliana and Xenopus
laevis (At64 and Xl64) and CraCRY using a spectroscopic electrochemical analysis called the xanthine/xanthine oxidase (X/XO) method29. In the well-established technique to evaluate the midpoint potentials of flavin
derivatives in flavoproteins, electrons released from the oxidation of X by XO simultaneously reduced FAD and
a reference dye in the presence of the redox mediator methylviologen (Fig. 1a). After testing various reference
dyes, we found that Safranin T dye was reduced simultaneously with FAD in At64, Xl64, and CraCRY. During
Figure 1. Evaluation of thermodynamic stabilities of F
ADH− in (6–4)PP-repairing proteins by the xanthine/
xanthine oxidase (X/XO) method. (a) The oxidation of X to uric acid by XO supplies electrons to FAD and
a reference dye Safranin T via a redox mediator methylviologen. The structural model was taken from the
reported crystal structure of At64 (PDB: 3FY4)32. (b) The X/XO reaction reduced fully oxidized FADox to twoelectron reduced anionic FADH− without accumulating one-electron reduced FADH·. (c) Midpoint potentials
of the FADox/FADH− redox pair in (6–4)PP-repairing proteins were ~ − 290 mV vs. SHE. The value was
considerably lower than those reported for CPD PL derived from Synechococcus elongatus (SePhrA), formerly
called Anacystis nidulans CPD P
L11,24 and for the solution FAD (aq) state33. Error bars for our samples reflect the
standard deviation for n = 3.
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the reduction by X/XO, no accumulation of the one-electron reduced F
ADH· state was observed, as confirmed by
probing the absorption unique to F
ADH· at wavelengths from 500 to 700 nm (Supplementary Fig. 1). The findings
indicated that the two-electron reduction of F
ADox into F
ADH− occurred in the system (Fig. 1b). Accordingly,
the synchronous decreases in the absorption characteristic to the oxidized states of FAD and Safranin T ( A450
for FADox, A510 for Safranin T in Supplementary Figs. 2–4) were analyzed using Eq. 1, as described in Methods. ...
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