Photoactivation Mechanism in BLUF Domains.
The observation of anionic and neutral semiquinones in the Slr1694
photocycle implies that light-driven ET is followed by PT between FAD
and aromatic residues in the protein. The picosecond time scale of
these events indicates that the residue(s) involved in the transfer
must be in close proximity to the isoalloxazine ring of the flavin.
Previous studies on flavo-enzymes have revealed that rapid light-driven
ET may occur from aromatic residues to flavin (25, 26).
Tyr-8 (Tyr-21 in AppA) is a good candidate because it is the closest
aromatic residue to the flavin and has been shown to be critical for
photocycling activity in BLUF domains (12, 15, 16).
The redox properties of flavin and Tyr gives a favorable driving force
for the ET reaction: the midpoint potential for flavin/flavin•− is approximately −0.8 V and for Tyr/Tyr•+ is ≈0.93 V (25, 27), providing a driving force for ET of ΔG = −0.62 eV given that the energy of the 0–0 transition of S0 → S1 is ≈ 2.35 eV (1 eV = 1.602 × 10−19 J) (27).
the various BLUF crystal structures, the dark-state orientation of the
conserved Gln remained ambiguous: according to Anderson et al. (11) the amino group of the conserved Gln would donate hydrogen bonds to N5 and the conserved Tyr, whereas Jung et al. (13) and Kita et al. (12)
favored an orientation where the Gln’s amino group donates a hydrogen
bond to O4 and receives a hydrogen bond from the conserved Tyr. Fourier
transform infrared results by Masuda et al. (17) indicated formation of hydrogen bonds to O4 and loss of a hydrogen bond to N5 of FAD upon illumination. Recent work by Unno et al. (28)
showed that mutation of the conserved Gln in the AppA BLUF domain did
not alter hydrogen bonding of O4. The latter observations are
consistent with a dark orientation as shown in Fig. 1A.
Thus, the accumulated evidence points favorably in the direction of a
hydrogen-bond switching by means of a Gln flip, as proposed by Anderson
et al. (11).
This mechanism also can explain why the photoactivated state is so
stable and lives from seconds in Slr1694 to a half-hour in AppA. In
support of this hypothesis, mutation of the conserved Tyr or the
conserved Gln results in a complete loss of photocycling activity (12, 15, 16, 28, 29). With the R. sphaeroides BlrB structure at hand, Jung et al. (13)
presented an alternative that would imply a light-induced PT from
Arg-32 to the O2 of the flavin from FAD*. This mechanism does not
involve radical flavin intermediates and is clearly at odds with the
We now are in a position to propose a
photochemical reaction mechanism for the photoactivation of BLUF
domains, which is schematically indicated in Fig. 5. In the dark state, the mutual orientation of Gln and FAD is arranged as indicated in Fig. 5A,
with hydrogen bonds from the amino group of Gln-50 to Tyr-8 and the N5
atom of FAD. Blue-light excitation induces a change in the redox
potential of FAD, which causes it to accept an electron, resulting in
formation of the FAD anionic semiquinone FAD•−, as indicated in Fig. 5B.
In view of the close proximity between FAD and the conserved Tyr, we
propose that Tyr-8 acts as the electron donor as suggested for AppA (30, 31).
This event breaks the hydrogen bond between Tyr-8 and Gln-50 as a
result of increased electrostatic repulsion by Tyr-8. The spectroscopic
properties of FAD•− as determined from the target analysis
indicate that it forms a CT complex, possibly with the oxidized Tyr-8
radical or perhaps with another positively charged residue in the
vicinity of FAD.
Upon its reduction, the flavin becomes a strong base that attracts a
proton. The closest proton source is the amino group of Gln-50, from
which most likely a proton is abstracted. In turn, Gln-50 abstracts a
proton from Tyr-8, which at the time is oxidized and highly reactive,
resulting in the radical pair FADH•–Tyr• (Fig. 5C).
The PT event takes 6 ps and slows down 3-fold upon deuteration. It is
well established that protonation of the neutral flavin semiquinone
radical FADH• takes place at N5 (32),
which inevitably breaks the hydrogen bond between N5 and Gln-50,
leaving Gln-50 unhinged and free to rotate. The hydrogen-bond rupture
constitutes the triggering event that allows an ≈180° flip of Gln-50,
which results in a new hydrogen bond from the amino group of Gln-50 to
the O4 of the flavin (Fig. 5D).
Possibly, the carbonyl of Gln-50 transiently accepts a hydrogen bond
from the newly protonated N5 (configuration not shown). After 65 ps,
radical-pair recombination takes place between FADH• and Tyr-8•,
whereby the hydrogen atom located on N5 of the flavin returns to Tyr-8,
possibly using the carbonyl of Gln-50 as a stepping stone (Fig. 5E).
The reprotonated Tyr then donates a hydrogen bond to the carbonyl group
of Gln-50, locking it in place. This final step is subject to a KIE of
2.4 upon H/D exchange and is attributed to formation of the long-lived
signaling state. In this conformation, the amino moiety of Gln-50
donates a hydrogen bond to O4 of the flavin, and the carbonyl moiety of
Gln-50 accepts a hydrogen bond from Tyr-8. Possibly, the amino moiety
of Gln-50 hydrogen bonds to N5 as well (configuration not shown). The
protein remains in this conformation for a long time because of high
The potential involvement of the conserved
Trp (Trp-91 in Slr1694, Trp-104 in AppA) is not taken into account
because earlier work has shown that Trp-104 is not required for
photocycling in AppA (20). The driving force is larger for Tyr than for Trp (net ΔG
= −0.62 eV for Tyr and −0.4 eV for Trp), and (from the AppA crystal
structure) the distances from Tyr-21 and Trp-104 to N5 of FAD are 4.5
and 5.7 Å, respectively (11), indicating that Tyr has a better spatial overlap with the flavin than Trp. The crystal structures of the R. sphaeroides BlrB and Thermosynechococcus Tll0078 BLUF domains indicated a different orientation for the conserved Trp, with a significantly increased distance to FAD (12, 13).
the present reaction model, predictions can be made with regard to the
photochemistry of the nonphotocycling Gln-50 and Tyr-8 mutants. With
Gln-50 deleted, ET and possibly PT may still occur from Tyr-8 upon
excitation of the flavin. In the absence of the switchable Gln-50
residue, the resulting radical pair will very likely recombine to the
original ground state without the production of long-lived
photoproducts. Upon deletion of Tyr-8, the light-driven ET process that
underlies the photoactivation reaction is shut off, which readily
explains the inability of mutants to photocycle. However, the conserved
Trp (Trp-91 in Slr1694, Trp-104 in AppA) may complement the ET process
to FAD without initiating the photocycle. Indeed, recent experiments on
the Tyr-21 mutant of AppA showed light-driven ET from Trp-104 on a
picosecond timescale, followed by radical-pair recombination on the
(sub)nanosecond timescale to the original ground state (ref. 31 and M.G., W. Laan, I.H.M.v.S., R.v.G., K. J. Hellingwerf, and J.T.M.K., unpublished data).
Comparison with Other BLUF Domains.
Given the similarities in structure and sequence between all BLUF
domains studied so far, a similar reaction mechanism very likely holds.
In AppA, the FAD* lifetime is >1 order of magnitude longer than in
Slr1694, with time constants of 90 and 590 ps. The red-shifted
signaling state is apparently formed directly from FAD* (18).
The reason that no transient reaction intermediates are observed
probably stems from a rate-limiting initial ET rate from FAD*. Such
differences in ET rates likely arise from different redox potentials of
FAD, because these are sensitive to its hydrogen-bonding pattern and
the vicinity of charged residues. It is interesting to note that in
AppA, a positively charged His-44 hydrogen bonds to the O2 in lieu of a
neutral Asn-31 in Slr1694. In the context of the present results, the
formation of the FAD•− anionic radical in AppA would occur with time constants of 90 and 590 ps, which rapidly evolves into the FADH• intermediate and long-lived product AppARED on much faster timescales. In this way, the FAD•− and FADH•
intermediates do not transiently accumulate during the reaction and
therefore escape detection. A rate-limiting light-driven ET rather than
a hydrogen transfer in the AppA BLUF domain is consistent with the
absence of a KIE on the FAD excited-state lifetime upon H/D exchange (20).
Light-Induced ET: A General Theme in Flavin-Binding Photoreceptors?
Three families of flavin-binding photoreceptors have thus far been
identified: the phototropins [with light, oxygen, or voltage (LOV)
domains as flavin-binding domains], the cryptochromes, and the BLUF
domains. Although their modes of action appear very distinct, there are
interesting parallels. The present results show that the hydrogen-bond
switch in BLUF domains is driven by initial ET from aromatic residues
to the oxidized flavin. In the cryptochromes, similar ET processes
appear to underlie their activity, with Trps as likely electron donors (10, 33).
The question remains whether in LOV domains, ET or PT from a conserved
Cys to the FMN chromophore constitutes the primary photochemical event (9, 34–36).
If the former situation applies, we may indeed conclude that it is the
redox potential of the flavin chromophore that defines the light
sensitivity of flavin-binding photoreceptors, very much so as it
defines the catalytic activity in most flavoenzymes (37).