Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor
contributions: R.v.G., P.H., and J.T.M.K. designed research; M.G.,
J.M.K., and D.C.L. performed research; P.H. contributed new
reagents/analytic tools; M.G., I.H.M.v.S., and J.T.M.K. analyzed data;
and M.G. and J.T.M.K. wrote the paper.
discovered class of photoreceptor proteins found in bacteria and
eukaryotic algae, where they control a range of physiological responses
including photosynthesis gene expression, photophobia, and negative
phototaxis. Other than in well known photoreceptors such as the
rhodopsins and phytochromes, BLUF domains are sensitive to light
through an oxidized flavin rather than an isomerizable cofactor. To
understand the physicochemical basis of BLUF domain photoactivation, we
have applied femtosecond transient absorption spectroscopy to the
Slr1694 BLUF domain of Synechocystis PCC6803. We show that
photoactivation of BLUF domains proceeds by means of a radical-pair
mechanism, driven by electron and proton transfer from the protein to
the flavin, resulting in the transient formation of anionic and neutral
flavin radical species that finally result in the long-lived signaling
state on a 100-ps timescale. A pronounced deuteration effect is
observed on the lifetimes of the intermediate radical species,
indicating that proton movements underlie their molecular
transformations. We propose a photoactivation mechanism that involves a
successive rupture of hydrogen bonds between a conserved tyrosine and
glutamine by light-induced electron transfer from tyrosine to flavin
and between the glutamine and flavin by subsequent protonation at
flavin N5. These events allow a reorientation of the conserved
glutamine, resulting in a switching of the hydrogen-bond network
connecting the chromophore to the protein, followed by radical-pair
recombination, which locks the glutamine in place. It is suggested that
the redox potential of flavin generally defines the light sensitivity
of flavin-binding photoreceptors.
information carried by light. Their responses are mediated by
photoreceptor proteins, which are sensitive to light through prosthetic
chromophore molecules. The past decade has witnessed the discovery of a
large number of novel flavin-binding photoreceptors, notably the
phototropins, the cryptochromes, and BLUF (blue-light sensing using
FAD) domains (1).
Phototropins are primarily found in plants and control several
physiological responses such as phototropism, chloroplast movement, and
stomatal opening (2), whereas cryptochromes are known to regulate growth and development in plants and circadian rhythms in plants and insects (3, 4).
BLUF domains are a distinct family of flavin-binding photoreceptors
that show no significant relationship to other sensor proteins in
sequence or structure. The BLUF domain was first discovered as the
N-terminal part of the flavoprotein AppA from the purple photosynthetic
bacterium Rhodobacter sphaeroides and was shown to control
photosynthesis gene expression in response to high-intensity blue-light
irradiation and variation of the oxygen tension (5, 6).
In later work, the BLUF-containing photoactivated adenylyl cyclase
(PAC) photoreceptor was demonstrated to mediate photophobic responses
in the green alga Euglena gracilis (7).
generation of a light signal by photoreceptor proteins relies on the
formation of a long-lived signaling state, where initial local
structural changes resulting from light absorption by the chromophore
are relayed to the protein surface, which undergoes specific
alterations that may be sensed by signaling partner proteins. In
“traditional” photoreceptors such as the rhodopsins, phytochromes and
the xanthopsins, the initial local change is achieved by rapid E/Z
isomerization photochemistry of their chromophore, which in turn
initiates larger conformational changes by creating steric conflicts,
or by charge movements among chromophore and apoprotein (1).
In the absence of isomerizable groups, flavin-binding photoreceptors
rely on different modes of light activation and thus provide an
alternative window on how photon absorption may be coupled to
biological sensory function. In the phototropins, for instance,
signaling occurs through the light-induced formation of a covalent
adduct between the flavin chromophore and a conserved cysteine in
light, oxygen, or voltage (LOV) domains, which eventually leads to
autophosphorylation of a C-terminal Ser/Thr kinase (8, 9).
In analogy with the related photolyases, cryptochromes are thought to
function by means of light-induced electron transfer (ET) reactions (10), but at present their mode of action remains largely obscure.
Recently, crystal and solution structures of several BLUF domains were obtained (11–14).
The BLUF domain shows a ferredoxin-like fold consisting of a
five-stranded β-sheet with two α-helices packed on one side of the
sheet, with the noncovalently bound isoalloxazine ring of flavin
adenine dinucleotide (FAD) positioned between the two α-helices. Fig. 1 shows a close-up of the FAD-binding pocket of the AppA BLUF domain (11).
FAD is involved in an extensive hydrogen-bond network with residues
lining the FAD binding pocket, including a highly conserved tyrosine
(Tyr) and glutamine (Gln). Upon illumination with blue light, BLUF
domains show a characteristic spectral red-shift of their absorption
spectrum by ≈10 nm, which is a unique feature of this photoreceptor
family and is thought to correspond to the signaling state (5).
light is under considerable debate. Early studies suggested that an
increased π–π stacking between Tyr and FAD, or deprotonation of FAD,
would induce signaling-state formation (15, 16). Jung et al. (13)
proposed that in the BlrB BLUF domain, proton transfer (PT) takes place
from an arginine (Arg) residue to the O2 atom of the isoalloxazine ring
upon blue-light absorption, triggered by the increased basicity of O2
upon promotion of FAD to the singlet excited state. Masuda et al. (17)
interpreted their results from Fourier transform infrared (FTIR)
spectroscopy by a hydrogen-bond rearrangement around the FAD
chromophore upon signaling-state formation. Anderson et al. (11)
have proposed a photoactivation model that involves such a
hydrogen-bond rearrangement accompanying a 180° rotation of the
conserved Gln, as shown in Fig. 1.
In this view, photon absorption leads to the breaking of hydrogen bonds
from the Gln amino group to the N5 of flavin and to the Tyr and
formation of a hydrogen bond to the O4 of the flavin. Ultrafast
spectroscopy on the AppA BLUF domain showed that the red-shifted
product state is formed in <1 ns, directly from the FAD singlet
excited state and without any apparent reaction intermediate (18), yielding no clues on the validity of any of these scenarios.
investigate the photochemistry in the Slr1694 BLUF domain of the
cyanobacterium Synechocystis PCC6803. We show that formation
of the red-shifted signaling state proceeds by means of a radical-pair
mechanism, driven by ET and PT processes to FAD from its protein
environment and resulting in the transient formation of anionic and
neutral FAD radical species. We propose a photoactivation mechanism
that involves a successive rupture of hydrogen bonds between the
conserved Tyr and Gln by ET and between the Gln and FAD by
light-induced protonation at N5, which allows a reorientation of the
conserved Gln and subsequent switch of hydrogen-bond network connecting
the chromophore to the protein.