Electron Transfer Quenching in Light Adapted and Mutant Forms of the AppA BLUF

Domain

Sergey P. Laptenok§, Andras Lukacs§,#, Richard Brust‡,┴, Allison Haigney‡,†, Agnieszka Gil‡,

Michael Towrie║, Gregory M. Greetham║, Peter J. Tonge‡* and Stephen R. Meech§*

‡Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400,

USA,

§School of Chemistry, University of East Anglia, Norwich NR4 7TJ, UK,

║Central Laser Facility, Research Complex at Harwell, Harwell Science and Innovation

Campus, Didcot, Oxon OX11 0QX, UK,

#Department of Biophysics, Medical School Szigeti str. 12#, H-7624 Pécs, Hungary

*Authors for correspondence (s.meech@uea.ac.uk; ptonge@notes.cc.sunysb.edu)

Present Address: ┴The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458

†University of Pennsylvania , Philadelphia, PA 19104 USA 

1

Abstract

The Blue Light Using Flavin (BLUF) domain proteins are an important family of

photoreceptors controlling a range of responses in a wide variety of organisms. The details of

the primary photochemical mechanism, by which light absorption in the isoalloxazine ring of

the flavin is converted into a structure change to form the signalling state of the protein, is

unresolved. In this work we apply ultrafast time resolved infra-red (TRIR) spectroscopy to

investigate the primary photophysics of the BLUF domain of the protein AppA (AppABLUF) a

light activated antirepressor. Here a number of mutations at Y21 and W104 in AppABLUF are

investigated. The Y21 mutants are known to be photoinactive, while W104 mutants show the

characteristic spectral red-shift associated with BLUF domain activity. Using TRIR we

observed separately the decay of the excited state and the recovery of the ground state. In

both cases the kinetics are found to be non-single exponential for all the proteins studied,

suggesting a range of ground state stuctures. In the Y21 mutants an intermediate state was

also observed, assigned to formation of the radical of the isoalloxazine (flavin) ring. The

electron donor is the W104 residue. In contrast, no radical intermediates were detected in the

studies of the photactive dark adapted proteins, dAppABLUF and the dW104 mutants,

suggesting a structure change in the Y21 mutants which favours W104 to isolloaxazine

electron transfer. In contrast, in the light adapted form of the proteins (lAppABLUF, lW104)) a

radical intermediate was detected and the kinetics were greatly accelerated. In this case the

electron donor was Y21 and major structural changes are associated with the enhanced

quenching. In AppABLUF and the seven mutants studied radical intermediates are readily

observed by TRIR spectroscopy, but there is no correlation with photoactivity. This suggests

that if a charge separated state has a role in the BLUF photocycle it is only as a very short

lived intermediate.

2

Introduction

A wide range of living organisms have evolved the means to detect light, either to exploit it

as a source of energy or to avoid its harmful effects. Consequently an array of photoreceptors

have been discovered, with perhaps the most famous being the rhodopsins, the photoreceptor

cells in the retina.1,2 These have been studied intensely for a number of years, allowing the

excited state reaction, an ultrafast isomerization, to be characterised in considerable detail.2,3

More recently a wide array of new photoreceptors has been discovered. Some, such as the

photoactive yellow protein, depend on an excited state structure change similar to the

photoisomerization in rhodopsin.4,5 However, in many others the blue light absorbing

isoalloaxazine ring of either flavin adenine dincucleotide (FAD) or flavin mononucleotide

(FMN) acts as the chromophore.6-9 In these cases the mechanism by which this planar

aromatic chromophore converts the energy of an absorbed photon into a structure change in

the surrounding protein matrix is incompletely understood. Thus, there is considerable

interest in determining the mechanism of operation of these flavoprotein based blue light

sensors. This fundamental interest has been further stimulated by the demonstration that these

proteins have applications in the emerging field of ‘optogenetics’ or, more generally,

‘optobiology’.10-12 In optobiology the light activated function of a protein is recruited by

genetically encoding it with a specific target. For example, light activated ion channel

rhodopsins were shown to mediate signalling in neuronal cells.13 More recently blue light

sensing flavoproteins have been used to introduce a range of light activated functionalities,

for example allowing optical excitation to modify protein-protein interactions or DNA

binding.11 In principle, this methodology affords the experimenter both spatial and temporal

control over a specific cellular response, and is thus a potentially powerful tool in cell

biology.

3

Three families of flavoprotein based photosensors have been identified: blue light using

flavin (BLUF) domains; light-oxygen-voltage (LOV) domains; cryptochromes and

photolyases.8,9 Collectively these are involved in controlling a wide variety of light activated

processes including phototaxis, stomatal opening, and circadian rhythms. Each family has

distinctive aspects to their light sensing mechanisms. For cryptochromes and photolyase,

which are found in plants, animals and fungi, there is strong evidence for radical

intermediates suggesting an electron transfer mechanism.14 The modular LOV domain unit,

found in a variety of proteins in organisms from plants to bacteria, exhibits a photochemical

mechanism which is mediated by the triplet state of the isoalloxazine ring, populated via

intersystem crossing.15,16 This triplet state reacts with an adjacent cysteine residue to form a

metastable adduct, which is sufficient to initiate a structure change in the protein, leading to

signalling state formation. The BLUF domain is also a modular unit found in bacterial

proteins, which operates by a third mechanism which is not yet fully resolved, and is the topic

of this investigation.

One of the best characterised BLUF domains is the light activated antirepressor protein AppA

found in Rhodobacter sphaeroides, where it is responsible for controlling the expression of

the photosynthetic apparatus in response to both light and oxygen levels.17-21 In this work we

focus on the isolated BLUF domain, AppABLUF. BLUF domain photoactivity is indicated by a

small (10 – 15 nm) red-shift in the electronic absorption spectrum of the isoalloxazine ring

on irradiation with light at around 450 nm.20 Thus, in both the dark state (dAppABLUF) and the

red-shifted light adapted signalling state (lAppABLUF) the flavin remains in its fully oxidized

form (the reduced forms such as radicals and ions have very different absorption spectra22).

The photoactivity is reversible, with the relaxation from lAppABLUF to dAppABLUF occurring

with a half-life of about thirty minutes, after which the protein can be re-excited.23,24

4

Time resolved studies showed that the red-shift develops within 1 ns of electronic excitation,

suggesting a role for ultrafast processes.25 However, recent time resolved IR measurements

on the femtosecond to millisecond time scale revealed that further evolution in the protein

structure occurs on multiple time scales, suggesting that the red shift is the start of a more

complex structure change.26 In addition, stationary state IR difference spectra showed that

the flavin carbonyl has a different H-bonding environment in lAppABLUF compared to

dAppABLUF, and that there was an associated change in the protein structure.27,28 Thus,

although the electronic structure of the flavin is unchanged between dAppABLUF and

lAppABLUF the H-bond environment is restructured on multiple time scales, from sub-

nanosecond to at least microseconds.

Crystal structures are an invaluable guide to mechanism. A structure of AppABLUF is shown in

Figure 1 along with the detail of the H-bonding environment of what is assumed to be

dAppABLUF.17 The published structures all suggest that residues Y21, Q63, N45, W104 and

M106 are important in the flavin binding pocket. Mutations to the conserved residues Y21

and Q63 show that they are essential for observation of the red-shift, and N45 is crucial to

flavin binding. Mutations in W104 are known to support the red-shift, but biological activity

is greatly reduced.29,30 A femtosecond to microsecond time resolved IR experiment showed

that in W104A the microsecond timescale light induced structure change in AppABLUF was

short circuited, implicating this residue in communicating the local structure change to the

protein.26

Unfortunately, there is some disagreement among the published crystal structures,

particularly with regard to the important residues Q63 and W104.17,31 The originally

proposed mechanism for signalling state formation was that Q63 isomerized between dark

and light adapted states giving rise to a change in H-bonding at the flavin carbonyl, in

agreement with IR difference spectra.17,32 However, other structures suggested a different

5

conformation for Q63 and a subtly different H-bonding environment, with W104 and M106

occupying different positions;31 these have been labelled the Win and Wout models. Associated

with this discussion has been the role of light induced structure changes in Q63, with

different models favouring either the original isomerization mechanism17,32 or an initial light

induced tautomerization.33,34 Two recent theoretical models favoured a Wout structure with a

tautomerization reaction in Q63 as the light activated trigger for structural change,35,36 while

another favoured Q63 rotation and a Win to Wout structure change.37 Interestingly Udvarhelyi

and Domratcheva recently suggested that the orientation of Q63 is not well defined in the

ground state, in which case light induced isomerization alone is unlikely to be the sole

structural trigger.38

A second point of debate concerns the driving force for the structure change. Gauden et al

proposed on the basis of transient absorption data that an excited state electron transfer

reaction between the conserved Y21 residue and the isoalloxazine ring forms a radical pair

state which undergoes proton transfer, giving rise to isomerization in Q63, and thus the

altered H-bond network seen in IR difference measurements.25,32,39 A transient radical state

was indeed observed in the BLUF protein PixD. However, early time resolved IR (TRIR)

measurements on dAppABLUF observed only an instantaneous perturbation to the protein H-

bond environment and no further kinetic intermediates on the nanosecond timescale.33 Such

an instantaneous perturbation was also demonstrated in the photoinactive Q63E mutant.40 As

a result it was proposed that optical excitation itself may be sufficient to perturb the H-bond

environment, and specifically that optical excitation might favour tautomerization in Q63.33

These data suggest that the role of the radical intermediates in the BLUF photocycle, which is

assumed to be important in a number of calculations, requires further clarification. Recently

Lukacs et al. presented a detailed study of the TRIR of three BLUF domains and contrasted

the results with model spectra of radical isoalloxazines and with flavoproteins which

6

unambiguously show electron transfer reactions.41 Although radical states are observed in

some cases there was no correlation between BLUF photoactivity and electron transfer.

Further, unnatural amino acid substitutions were used to artificially modulate the redox

potential of the putative Y21 donor, but the results were not consistent with a simple electron

transfer reaction.

In this work we extend our TRIR studies of the AppABLUF domain to a number of mutants of

Y21 and W104 and seek a correlation between the observability of radical intermediates, the

rate of reaction and photoactivity. Radical intermediates are observed in a number of cases

involving both W104 and Y21 as donors, but no correlation is observed between their rate of

formation and photoactivity. The results are discussed in terms of the local structure and the

environment of the isoalloxazine chromophore.

Experimental

Protein Expression and Purification: Site-directed mutagenesis on AppABLUF in a pET-15b

vector was performed using pfuTurbo(Agilent). Primer sequences for W104A were: forward:

5’-TTTGCGGGAGCGCACATGCAGC TCTCCTGCTCG-3’, reverse 5’

CGAGCAGGAGAGCTGCATGTGCGCTCCCGCAAA-3’. Primer sequences for Y21C

were 5’ CTG GTT TCC TGC TGC TGC CGC AGC CTG GCG GCC 3’ (Forward) and 5’

GGC CGC CAG GCT GCG GCA GCA GCA GGA AAC CAG 3’ (Backward); for Y21S

were 5’ CTG GTT TCC TGC TGC AGC CGC AGC CTG GCG GCC 3’ (Forward) and 5’

GGC CGC CAG GCT GCG GCT GCA GCA GGA AAC CAG 3’ (Backward); for Y21I

were 5’ CTG GTT TCC TGC TGC ATC CGC AGC CTG GCG GCC 3’ (Forward) and 5’

GGC CGC CAG GCT GCG CAT GCA GCA GGA AAC CAG 3’ (Backward). All

constructs were verified by DNA sequencing. Plasmids were transformed into BL21(DE3) E.

coli cells. A single colony was used to inoculate 10 mL starter cultures containing LB media

7

with 200 μg/mL ampicillin at 37°C which was grown overnight and used the following day to

inoculate a 1 L LB/ampicillin culture. Cells were grown to an OD600 of about 1 at 30°C for 5

hours. Induction was performed by adding 0.8 mM IPTG at 18°C, followed by overnight

incubation in the dark. The following day cells were harvested by centrifugation at 5000 rpm

at 4 °C and stored at -20 °C until needed.

Purification of wild type and the various mutants were performed in the dark. Thawed cell

pellets were resuspended in 40 mL of wash buffer (10 mM NaCl, 50 mM Na2H2PO4, pH

8.0), to which 200 μL of 50 mM PMSF was added to inhibit endogenous protease activity.

Cells were lysed by sonication and cell debris was removed by ultracentrifugation (33000

rpm for 90 minutes). The cell pellet was discarded, and to the soluble fraction 1 mL of a

10mg/mL solution of FAD was added to the supernatant and incubated for 45 minutes on ice.

The protein was loaded onto an equilibrated Ni-NTA affinity column. The bound protein was

then washed using increasing amounts of imidazole and finally eluted with 250 mM

imidazole. The fractions were then dialyzed overnight in the dark in the 3L of wash buffer.

Protein purity and yield were determined using UV-Vis spectroscopy and SDS-PAGE.

Ultrafast Measurements All measurements reported here used the 10 kHz ULTRA facility

developed at the Central Laser facility of the Rutherford Appleton Laboratories which are

described in detail elsewhere.42,43 ULTRA offers wide tunability in the visible region, a broad

bandwidth in the IR probe, sub 100 fs rime resolution and excellent stability. In the present

experiments it was used in the visible pump – IR probe geometry. The excitation (pump)

wavelength was 450 nm with a pulse energy of a few hundred nJ focussed to a 200 micron

spot size. It was checked that the spectra and kinetics were independent of the pump

wavelength and pulse energy. IR transmission was measured sequentially for pump-on and

pump-off using a 5 kHz mechanical chopper, and the data processed to provide the TRIR

difference spectra as described elsewhere.

8

Many dark adapted BLUF domains exhibit a light to dark recovery time of several minutes.

Thus, to obtain transient data of the dark state the sample has to be both rastered in the beam,

so that the sample is not subject to multiple excitations, and flowed so that fresh sample is

present after each cycle of the raster. In the present case ca 1 ml of 1 mM protein solution in

phosphate buffer made up in D2O at pD 8 was circulated through a 50 micron path length

CaF2 cell. For the study of light adapted states the sample was not flowed and was irradiated

for 3-4 min. at 385 nm then held in the beam under continuous irradiation during the

measurement.

Data Analysis Data were assessed in two ways. First, we are interested in the formation (or

otherwise) of intermediate states in the ultrafast kinetics. Using TRIR we have previously

identified marker modes for the excited state decay and ground state recovery.41 Thus these

can be fit separately, and any difference between them suggests the formation of a metastable

intermediate, which should also be visible in TRIR spectra (for example in the case of

electron transfer, the radical state of the isoalloxazine ring). Thus transient data at the two

characteristic wavenumbers were analysed separately. It was found that the kinetics were

non-single exponential in all cases but could be fit with a bi-exponential function with fast

and slow components, suggesting an inhomogeneous system; including further exponentials

in the fit gave a statistically better description but with increasing errors. Thus all marker

mode data were fit to the bi-exponential function and the separate decay times are reported,

along with their weighted average for the mean decay time. Second, we have conducted a

global analysis of the complete data set using established methods.44 Initially simple

sequential kinetic models were assumed, giving evolution associated difference spectra

(EADS). However, these simple models do not reproduce the non-single exponential

relaxation. Thus, in some cases specific target models were assumed in an effort to represent

9

the underlying processes, this analysis results in species associated difference spectra

(SADS).

Results and Discussion

Y21 is essential to photoactivity in AppA and PixD, and exchanging it for any other residue

suppresses the light induced red-shift in the flavin absorption. The W104 residue appears to

have a role in transmitting the primary events of optical excitation to the rest of the protein to

form the signalling state. Mutations at W104 in AppABLUF result in a protein which does

exhibit a red-shift in the flavin spectrum, but recovery of the dark state is accelerated, and

biological activity correspondingly attenuated. Our recent TRIR studies suggest that this

arises because the primary events in the photocycle occur, but these are not communicated to

the rest of the protein.26 Here we report TRIR data for the single AppA mutants Y21S, Y21I,

Y21C, W104Y, W104T and W104A and for the double mutant Y21I/W104A; Gauden et al

previously studied a similar series using transient absorption.45 As expected, all W104

mutants showed a red-shift under blue light irradiation while Y21 mutants did not. However,

the TRIR spectra reveal a variety of excited state chemistry.

Photoinduced Electron Transfer in Y21 Mutants TRIR spectra for Y21S recorded one ps

after excitation are compared with those for dAppABLUF in Figure 2. In TRIR spectra the

negative OD peaks appearing immediately upon excitation (bleaches) can be assigned to

modes of the ground state whose populations have been reduced by electronic excitation.

These modes may be localised either in the chromophore or the surrounding protein matrix; it

was shown elsewhere that excitation of the isoalloxazine ring can instantaneously perturb the

local protein H-bond environment.40 The highest frequency bleach in dAppABLUF (ca 1701 cm-

1) was assigned to a mainly C4=O localised mode (although C4=O and C2=O modes are

coupled, with the degree of coupling depending on the environment46). In Y21S (Figure 2),

10

and all the other Y21 mutants studied, the frequency of C4=O mode is unchanged on

mutation, suggesting that although photoinactive, the H-bonding environment of the

isoalloxazine ring remains similar to that in dAppABLUF. Note also that the transient

absorption signal (positive OD) seen at 1666 cm-1 in dAppABLUF is suppressed in Y21S

(Figure 2) and the other Y21 mutants. As previously noted33 this instantaneously formed

bleach acts as a marker mode for photoactivity, being absent or obscured in photoinactive

mutants of AppABLUF, which is also the case here.

The temporal evolution of the TRIR for Y21S is shown in Figure 3a, and is representative of

other Y21 mutants. Most significantly a transient absorption 1531 cm-1 is seen to grow in

with time, reaching its maximum OD in several tens of picoseconds; this wavenumber was

shown in model studies to be associated with an IR active vibrational absorption of the flavin

radical.47 Thus, the appearance of this mode suggests that these photoinactive mutants support

an electron transfer reaction. The same result was found earlier by Gauden et al for Y21F,

Y21I and Y21C in transient absorption.45 However, the vibrational resolution of TRIR allows

for a clearer separation of transient species, including in this case the identification of the

isoalloxazine radical absorption. Further the decay of the singlet excited state and the

recovery of the ground state can be monitored separately in TRIR, from the decay of the 1380

cm-1 transient absorption and the recovery of the 1548 cm-1 ground state bleach respectively.

As expected for the formation of an intermediate the excited state decays more rapidly than

the ground state recovers (Table 1). However, the difference between the two is quite small,

suggesting that once the radical is formed it decays quite quickly to the ground state. To

illustrate this behaviour time resolved data for the 1380 cm-1 decay and the 1548 cm-1 ground

state recovery of Y21S are compared in Figure 3b. The separation between the two is

apparent, whereas for dAppABLUF the traces overlapped,41 suggesting the excited state decay

11

and ground state recovery match, consistent with a short lived (or absent) intermediate in

dAppABLUF.

The Y21 mutant data can be globally analysed assuming an FAD* FAD•− FAD kinetics

scheme. The data were fit to this simplest possible model, which assumes that the excited

state decays can be represented approximately by a single exponential decay function, which

is not the case; the non-single exponential kinetic introduced considerable ambiguity into the

analysis. Nevertheless, the fit to the data by the simplest model is reasonable, with the singlet

decaying to the intermediate in 31 ps which goes to a final state in 400 ps; the EADS are

shown in Figure 4a. In this case the 1531 cm-1 transient absorption associated with the FAD•−

appears in the spectrum of the intermediate, consistent with the scheme. However, the 1380

cm-1 transient associated with FAD* persists for longer times than the radical formation,

which is unphysical, but is a consequence of imposing a simple single exponential decay

scheme on non-single exponential data. Note also that the final state has finite amplitude

suggesting some long lived modification to the spectrum in Y21 mutants. This could be a

change in the H-bond environment of the FAD or in principle a long lived triplet state

(although the triplet state of FMN does not have any contribution around 1630 cm-1, which is

an important part of the final spectrum, Figure 4a26). In an effort to model these inherently

non-single exponential data they were analysed assuming two initial states (A and A’), one of

which decays to the radical (A•−) and one directly to the ground state; the condition that the

1380 cm-1 peak associated with FAD* is absent in the FAD•− spectrum was also imposed. The

radical state then relaxes to the final state (B), which is again slightly perturbed compared to

the ground state. The scheme is shown in Scheme 1 and the SADS and relaxation times in

Figure 4b.

12

Scheme 1

Again the fit is good, with the radical mainly associated with the intermediate A•−, the decay

of A and A’ reproducing the nonexponential decay at 1380 cm-1 and the extended A•− decay

giving rise to the slower recovery at 1548 cm-1. However, this scheme is not unique, many

others could be tried, and even with the high signal-to-noise presently available (Figure 3) the

data are not sufficient to distinguish between them. This simple scheme 1 is interesting

because it raises the possibility of an inhomogeneous ground state with one population able to

undergo radical formation and one which cannot. This is consistent with the non-exponential

decay of FAD* and also with recent calculations which found (for the dAppABLUF structure)

that Q63 could occupy different conformers.38 It is plausible that each conformer has

associated with it a re-organised H-bond structure, with each of the various orientations

having distinct photochemistry.

Concerning the identity of the electron donor, in the absence of Y21 the most likely candidate

is the adjacent tryptophan residue W104.45 In line with this, the double mutant Y21I /W104F

did not show any radical like absorption at 1531 cm-1. The previously studied (photoinactive)

mutant Y21W also showed rapid and efficient electron transfer and clear intermediate state

kinetics; thus, in that case Trp is unambiguously the electron donor.41 The rate of electron

transfer in Y21W was much higher than for the present Y21 mutants, which is consistent with

W21 being located close to the isoalloxazine ring. Interestingly along with the expected

radical isoalloxazine feature at 1531 cm-1, Y21W showed an additional intense sigmoidal

transient/bleach feature about 1630 cm-1 in both the experimental spectra and the EADS

recovered from global analysis.41 This 1630 cm-1 feature is also evident in the EADS of Y21S

13

(Figure 4a) and other Y21 mutants, but is of lower amplitude than for Y21W. Thus, these

data are consistent with Trp being the electron donor in both cases, and the difference in the

intermediate spectra suggests that W104 and W21 occupy different H-bond environments,

with different amplitude vibrational modes.

Photodynamics in dW104 Mutants These mutants all showed a red-shift on irradiation at

450 nm, a necessary (but not sufficient29) condition for photoactivity. However, the signalling

state (light) to dark relaxation was accelerated compared to dAppABLUF, by a factor which

varies between three for W104M and greater than twenty for W104A. The TRIR spectrum

recorded shortly after excitation of dW104T is shown in Figure 5a and compared with that

for dAppABLUF. As was observed for the Y21 mutants (Figure 3) there are only small effects

of W104 mutation on the spectra when compared to dAppABLUF. The C4=O mode is shifted

slightly to higher wavenumber (by 3 cm-1, which is close to our instrumental resolution, but a

similar shift was seen by Masuda and co-workers29) while the 1666 cm-1 photoactive marker

mode is retained. This is consistent with a dAppABLUF rather than a lAppABLUF environment

(see below) for these mutants.

The temporal evolution for W104T is shown in Figure 5b, and is representative of the other

W104 mutants. In contrast to what was observed for the photoinactive Y21 mutants, the

photoactive (as judged by red-shift under irradiation) W104 mutants do not show the

appearance of a flavin radical peak in the 1531 cm-1 region, a result which they share with the

photoactive dAppABLUF33 and BlsA48 (though not PixD32,39,41). Further, as was also found for

these photoactive BLUF domains,41 the decay of the excited state (1380 cm-1) matches the

recovery of the ground state (1548 cm-1), which is again inconsistent with a long lived charge

separated intermediate (Table 1). Thus, there is no obvious correlation between the

observation of an intermediate radical state in the BLUF photocycle and BLUF photoactivity;

in fact the mutations studied here suggest an anti-correlation, with the flavin radical

14

intermediate only being observed in photoinactive Y21 mutants. The absence of a radical

feature in the photoactive W104 mutants thus requires that either the Y21-FAD

Y21•+FAD•− electron transfer reaction does not occur, or that that the decay of the radical state

once formed is accelerated compared to Y21, such that the concentration is very low. This

point is discussed further below.

Radical Intermediates in lAppABLUF and lW104 mutants

The light adapted form lAppABLUF is readily generated by irradiation and persists for some

time because of its slow light to dark recovery. For lW104 mutants the recovery is faster, but

they may also be studied if the sample is continuously irradiated. Thus, TRIR of the

metastable light adapted states can be measured (Figure 6a). Two features are immediately

apparent. First, as noted previously,25,33,49 the excited state decay and ground state recovery

are >10 times faster than for dAppABLUF, and second that the electron transfer reaction

proceeds quite efficiently in lAppABLUF, as indicated by the transient formation of the flavin

radical intermediate (here at 1525 cm-1) and the much faster decay at 1380 cm-1 compared to

the 1548 cm-1 recovery (Table 2, Figure 6b). The changes in the bleach spectrum between the

TRIR of d and lAppABLUF (comparing with the data in Figures 2 and 5) were described

elsewhere, and can be understood on the basis of the modified H-bonding around the

isoalloxazine ring on formation of the light adapted state, with the red-shift in the C4=O

ground state bleach aligning with the light minus dark steady state IR difference spectra

reported by Masuda and co-workers.29,50

The complex lineshape of the TRIR spectra in the 1510 – 1530 cm-1 region for lAppABLUF

(Figure 6a) suggests a fast evolution of the spectral profile in the flavin radical region, which

is different to the behaviour seen in the Y21 mutants. This is highlighted by the global

analysis of lAppABLUF (Figure 6b), in which a simple sequential model was assumed (i.e.

15

again imposing single exponential decay on FAD*) but with a second intermediate. In this

model the excited state decays to generate a transient species around 1520 cm-1 which

subsequently evolves to yield a second transient species at 1530 cm-1. This may be consistent

with a fast ground state proton transfer reaction FAD•− FADH• following excited state

electron transfer. However, changes in H-bonding to the protein could also contribute to the

observed spectral shift; unfortunately the available model spectra47,51 are not sufficiently

detailed to resolve the spectrum further. The second intermediate essentially repopulates the

ground state without leaving any long lived structural perturbation (unlike Y21 mutants).

The enhanced quenching in lAppABLUF must arise from a reorganisation of the protein in the

light adapted state to dramatically enhance electron transfer. One possibility for lAppABLUF is

that W104 moves from an ‘out’ to an ‘in’ configuration, and this gives rise to fast excited

state quenching, the driving force for a Trp donor being favourable.52 However, the mutant

data are inconsistent with this assignment. All the lW104A lW104T and lW104Y mutants

also show faster excited state decay and ground state recovery than their dark adapted states,

the effect being noticeable in the average decay time and particularly in the slow component

of the decay/recovery. In addition each shows the formation of radical intermediate states.

These mutant data show that the likely electron donor in the light adapted state is Y21.

Further support for this is obtained from our studies with unnatural amino acid substitution,

where modulation of the redox potential of Y21 through substitution with fluorinated tyrosine

modifies the kinetics in the light adapted state (unpublished). Significantly, the formation of

observable radical intermediates is only readily detected in a photo-inactivated forms of the

protein, in this case lAppABLUF, but is absent in dAppABLUF.

Relation of AppA Structure to Quenching

The classical Marcus expression for the rate constant of an electron transfer reaction, ket, is:53

16

k et=2 π ⟨V el⟩

2

ℏ (4 πλ kT )1/2 exp−[ ( ΔG0+ λ )2

4 λ kT ],

in which G0 is the free energy change associated with the reaction, which is related to the

reduction and oxidation potentials of the reactants, Vel is the electronic coupling parameter

between donor and acceptor and the reorganization energy. Both of the latter parameters

depend on the details of the local structure and environment, while is also a function of

G0. The other parameters have their usual meaning. This equation defines the Marcus curve,

a parabola with maximum at –G0 = separating the normal and Marcus inverted regions.

As discussed by Lukacs et al, the failure to observe a radical intermediate following

photoexcitation of the isoalloaxazine ring in dAppABLUF, an observation which is repeated

here for the photoactive dW104 mutants, may be explained by charge recombination (CR)

occurring faster than charge separation (CS) in the FAD*-Y21 pair.41 With the available

information on ΔG0CR

and ΔG0CS

this suggested a reaction in the normal region of the Marcus

parabola. However, modulation of ΔG0CS/CR

, achieved by replacing Y21 with fluorinated

tyrosine derivatives, did not result in the expected acceleration of the excited state decay at

1380 cm-1.41 Thus for dAppABLUF (and, by extension, for the dW104 mutants) there is no

conclusive experimental evidence that charge separation is actually the driving force for

BLUF photoactivation. What is clear is that electron transfer accompanied by formation of a

radical intermediate state is possible in a number of cases (lAppA, the lW104 mutants, the

Y21 mutants) in which the BLUF photoactivation pathway is suppressed. In these cases both

Y21 (in the lW104 mutants) and W104 (in the Y21 mutants) have been shown to act as the

electron donor, where electron transfer was inferred from observations of a transient

absorption at a frequency associated with the isoalloxazine radical. This is turn suggests that

in these cases the charge separation reaction occurs in the Marcus inverted region, which

17

could arise from a change in the reorganization energy or the driving force by a few hundred

meV.

For the Y21 mutants, the electron donor is proposed to be W104 and the excited state

decay/ground state recovery times are quite similar to that of dAppABLUF. That the radical

intermediate is not seen in dAppABLUF suggests either that the electron transfer does not occur

or that the CS state is much shorter lived compared with the Y21 mutants. In either case, this

implies a significant change in the protein environment (compared to dAppABLUF) around

W104/FAD in the Y21 mutants. In contrast to the Y21 mutants the ‘photoactive’ (i.e. red-

shifting) dW104 mutants show no intermediate, although they also have similar spectra and

decay kinetics to dAppABLUF. The change in biological activity for these mutants probably

arises from their inability to communicate the photoinduced change in the local structure to

the remainder of the protein.26 In lAppABLUF and in the lW104 mutants the excited state

kinetics are greatly accelerated and an intermediate assigned to a radical state is observed.

This more efficient quenching (which has been noted before) indicates a substantial change in

the excited state chemistry. In the most extreme case this could arise from a change in the

charge state of the reactants (e.g. from tyrosine to tyrosinate), thus modifying G0.

Alternatively, the change in structure associated with formation of the light adapted state

must significantly alter the electronic coupling as well as the reorganization energy.

The question which is not really addressed by these considerations is the origin of the

nonexponential kinetics in all BLUF proteins so far studied. This observation suggests that a

distribution of ground state structures exists in solution in thermal equilibrium, and that each

structure has a different excited state chemistry, with perhaps only the minor populations

being photoactive. Recent calculations indeed suggested a distribution of Q63 rotamers.38 A

similar distribution may also exist in the excited state. It is then possible to imagine a

fluctuation in the structure in one of the conformers about FAD* leading to a reactive

18

conformation. In this conformation the reaction (be it tautomerization, electron or proton

transfer, or some combination thereof) must occur on an ultrafast timescale returning the

protein directly to a ground electronic state which, on further relaxation, traps the protein in

its light adapted state, characterized by a reorganized H-bond network around FAD. Such a

distribution would further complicate the analysis of BLUF domain photoactivity, as the

distribution itself might also be sensitive to interactions between the protein and its complex

partners. Thus investigation of these complexes is an important goal.

Summary

The photodynamics of a number of Y21 and W104 mutants of the dAppABLUF domain have

been studied by ultrafast TRIR spectroscopy. In all cases excited state decay and ground state

recovery could be monitored by the study of distinct marker modes. In every case the kinetics

departed from simple single exponential behavior, which is assigned to a distribution of

ground states structures. In the cases where the light induced red-shift of the isoalloxazine

absorption occurs, which indicates the onset of photoactivity in BLUF domains (i.e. in

dAppABLUF and the dW104 mutants) no intermediate states could be detected in TRIR

spectra. In contrast, in the photoinactive Y21 mutants and in the light adapted lAppABLUF and

lW104 mutants an intermediate could be observed, with an absorption associated with the

isoalloxazine radical anion. In the Y21 mutants the donor is likely to be W104 and in the light

adapted forms it is Y21. There is no indication in these measurements for formation of a

metastable charge separated state in the BLUF photocycle. Finally, the efficient quenching in

the light adapted states suggests a very significant change in the local structure or in the

reactants of the Y21/FAD pair.

19

Acknowledgements We are grateful to EPSRC and NSF for financial support of this project

(EP/K000764/1 to SRM, CHE-1223819 to PJT) and to STFC for programme access to the

Central Laser Facility. AL is a Bolyai Janos Research Fellow and was supported by OTKA

NN113090.

20

Figure Legends

Figure 1. (a) Structure of AppABLUF determined by crystallography (PDB 1YRX). (b) Details

of the H-bonding environment around the isoalloxazine ring of FAD. Note that alternative

structures exist which differ on the orientation and location of Q63 and W104/M106 (PDB

2IYG – see text).

Figure 2 TRIR spectra for dAppABLUF and Y21S recorded straight after electronic excitation.

Negative OD signals correspond to bleaching of ground state vibrational modes, whereas

positive OD indicates promptly formed transients.

Figure 3 (a) TRIR spectra of Y21S measured as a function of time after excitation. Note the

appearance over tens of picoseconds of the transient absorption around 1530 cm-1. (b)

Transient data for Y21S at 1380 cm-1 and (inverted for comparison) 1548 cm-1; also shown

are the intermediate kinetics at 1531 cm-1.

Figure 4 (a) EADS from analysis of the Y21S data (Figure 3a) using a sequential kinetic

scheme with a single intermediate (b) SADS associated with Scheme 1 (see text). Timescales

associated with each step are shown. Note the long lived final structure consistent with a

metastable light induced change in the protein structure.

Figure 5 (a) TRIR of W104T compared with dAppABLUF immediately after excitation (b)

Time dependence of TRIR for W104T.

Figure 6 (a) Evolution of TRIR for lAppABLUF (b) EADS for lAppABLUF recovered from a

sequential analysis assuming two intermediate states. Note that the final spectrum has

essentially zero amplitude suggesting repopulation of the ground state.

21

Figure 1

Figure 2

22

a

b

Figure 3

23

Figure 4

24

Figure 5

25

Figure 6

26

Table 1. Excited state decay (1380 cm-1) and ground state recovery (1548 cm-1) kinetics for

dAppA, dW104 and Y21 mutants. Tau 1 and 2 were recovered from a biexponential fit to the

data and the weight is the amplitude of the fast component. A constant offset was included,

and has a significant amplitude in the 1548 cm-1 recovery (Figures 3,5).

BLUF Frequency/cm-1 Weight tau 1 Tau1/ps Tau2/ps <Tau>/ps

dAppA 1380 0.38 17±8 401±110 255

1548 0.42 29±5 512±60 309

Y21S 1380 0.48 25±7 350±100 194

1548 0.36 38±10 413±70 278

Y21C 1380 0.71 49±20 420±inf 156

1548 0.41 50±13 431±90 274

Y21I 1380 0.56 26±7 313±105 152

1548 0.48 48±9 431±98 247

Y21IW104F 1380 0.32 7±4 152±30 105

1548 0.53 81±30 294±120 181

W104A 1380 0.37 4±3 663±290 419

1548 0.42 21±6 598±140 355

W104T 1380 0.27 4±inf 300±85 219

1548 0.28 20±4 465±40 340

W104Y 1380 0.27 9±3 488±70 358

1548 0.27 11±3 478±45 351

27

Table 2. Excited state decay (1380 cm-1) and ground state recovery (1548 cm-1) kinetics for

lAppABLUF and lW104 mutants

Frequency/cm-1 Weight 1 1/ps 2/ps <Tau>/ps

lAppABLUF 1380 0.26 1.4(±1) 21(±3) 15

1548 0.77 16.3(±1) 190(±40) 56

lW104A 1380 0.89 1±inf 16±inf 2

1548 0.8 17±9 474±1inf 108

lW104T 1380 0.71 8±4 108±70 37

1548 0.63 6±91 102±15 41

lW104Y 1380 0.65 7±3 93±40 37

1548 0.67 9±1 161±25 59

28

References

1 Smith, S. O. in Annual Review of Biophysics, Vol 39 Vol. 39 Annual Review of Biophysics (eds D. C. Rees, K. A. Dill, & J. R. Williamson) 309-328 (2010).

2 Wand, A., Gdor, I., Zhu, J. Y., Sheves, M. & Ruhman, S. Shedding New Light on Retinal Protein Photochemistry. Annual Review of Physical Chemistry, Vol 64 64, 437-458, doi:10.1146/annurev-physchem-040412-110148 (2013).

3 Wand, A., Friedman, N., Sheves, M. & Ruhman, S. Ultrafast Photochemistry of Light-Adapted and Dark-Adapted Bacteriorhodopsin: Effects of the Initial Retinal Configuration. Journal of Physical Chemistry B 116, 10444-10452, doi:10.1021/jp2125284 (2012).

4 Schotte, F. et al. Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography. Proc. Natl. Acad. Sci. U. S. A. 109, 19256-19261, doi:10.1073/pnas.1210938109 (2012).

5 Kim, T. W. et al. Protein Structural Dynamics of Photoactive Yellow Protein in Solution Revealed by Pump-Probe X-ray Solution Scattering. Journal of the American Chemical Society 134, 3145-3153, doi:10.1021/ja210435n (2012).

6 Masuda, S. Light Detection and Signal Transduction in the BLUF Photoreceptors. Plant and Cell Physiology 54, 171-179, doi:10.1093/pcp/pcs173 (2013).

7 Losi, A. Flavin-based blue-light photosensors: A photobiophysics update. Photochemistry and Photobiology 83, 1283-1300, doi:10.1111/j.1751-1097.2007.00196.x (2007).

8 Losi, A. & Gartner, W. Old Chromophores, New Photoactivation Paradigms, Trendy Applications: Flavins in Blue Light-Sensing Photoreceptors. Photochemistry and Photobiology 87, 491-510, doi:10.1111/j.1751-1097.2011.00913.x (2010).

9 Conrad, K. S., Manahan, C. C. & Crane, B. R. Photochemistry of flavoprotein light sensors. Nat Chem Biol 10, 801-809, doi:10.1038/nchembio.1633 (2014).

10 Deisseroth, K. Optogenetics. Nature Methods 8, 26-29, doi:10.1038/nmeth.f.324 (2011).11 Kim, B. & Lin, M. Z. Optobiology: optical control of biological processes via protein

engineering. Biochemical Society Transactions 41, 1183-1188, doi:10.1042/bst20130150 (2013).

12 Moglich, A., Yang, X. J., Ayers, R. A. & Moffat, K. in Annual Review of Plant Biology, Vol 61 Vol. 61 Annual Review of Plant Biology (eds S. Merchant, W. R. Briggs, & D. Ort) 21-47 (Annual Reviews, 2010).

13 Hegemann, P. & Moglich, A. Channelrhodopsin engineering and exploration of new optogenetic tools. Nat Meth 8, 39-42.

14 Lukacs, A., Eker, A. P. M., Byrdin, M., Brettel, K. & Vos, M. H. in Ultrafast Phenomena Xvi Vol. 92 Springer Series in Chemical Physics (eds P. Corkum, S. DeSilvestri, K. A. Nelson, & E. Riedle) 604-606 (2009).

15 Raffelberg, S., Mansurova, M., Gärtner, W. & Losi, A. Modulation of the Photocycle of a LOV Domain Photoreceptor by the Hydrogen-Bonding Network. Journal of the American Chemical Society 133, 5346-5356, doi:10.1021/ja1097379 (2011).

16 Bednarz, T., Losi, A., Gartner, W., Hegemann, P. & Heberle, J. Functional variations among LOV domains as revealed by FT-IR difference spectroscopy. Photochemical & Photobiological Sciences 3, 575-579, doi:10.1039/b400976b (2004).

17 Anderson, S. et al. Structure of a novel photoreceptor, the BLUF domain of AppA from Rhodobacter sphaeroides. Biochemistry 44, 7998-8005 (2005).

18 Dragnea, V. et al. Crystallographic and functional studies of the AppA blue-light receptor BLUF domain from Rhodobacter sphaeroides. Abstracts of Papers of the American Chemical Society 230, U2809-U2809 (2005).

19 Masuda, S. Molecular mechanism of light-dependent signal transduction pathway in AppA. Plant and Cell Physiology 47, S7-S7 (2006).

29

20 Masuda, S. & Bauer, C. E. AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides. Cell 110, 613-623 (2002).

21 Masuda, S., Hasegawa, K. & Ono, T. Light-induced structural changes of apo-protein and chromophore in the BLUF domain of AppA for a signaling state. Plant and Cell Physiology 46, S103-S103 (2005).

22 Massey, V. The chemical and biological versatility of riboflavin. Biochemical Society Transactions 28, 283-296 (2000).

23 Dragnea, V., Arunkumar, A. I., Lee, C. W., Giedroc, D. P. & Bauer, C. E. A Q63E Rhodobacter sphaeroides AppA BLUF Domain Mutant Is Locked in a Pseudo-Light-Excited Signaling State. Biochemistry 49, 10682-10690, doi:10.1021/bi1002162 (2010).

24 Laan, W. et al. On the mechanism of activation of the BLUF domain of AppA. Biochemistry 45, 51-60 (2006).

25 Gauden, M. et al. Photocycle of the flavin-binding photoreceptor AppA, a bacterial transcriptional antirepressor of photosynthesis genes. Biochemistry 44, 3653-3662 (2005).

26 Brust, R. et al. Proteins in Action: Femtosecond to Millisecond Structural Dynamics of a Photoactive Flavoprotein. Journal of the American Chemical Society 135, 16168-16174, doi:10.1021/ja407265p (2013).

27 Masuda, S., Hasegawa, K., Ishii, A. & Ono, T. Light-induced structural changes in a putative blue-light receptor with a novel FAD binding fold sensor of blue-light using FAD (BLUF); Slr1694 of Synechocystis sp PCC6803. Biochemistry 43, 5304-5313 (2004).

28 Masuda, S., Hasegawa, K. & Ono, T. Light-induced structural changes of apoprotein and chromophore in the sensor of blue light using FAD (BLUF) domain of AppA for a signaling state. Biochemistry 44, 1215-1224 (2005).

29 Masuda, S., Hasegawa, K. & Ono, T. A. Tryptophan at position 104 is involved in transforming light signal into changes of beta-sheet structure for the signaling state in the BLUF domain of AppA. Plant and Cell Physiology 46, 1894-1901 (2005).

30 Masuda, S., Tomida, Y., Ohta, H. & Takamiya, K. I. The critical role of a hydrogen bond between Gln63 and Trp104 in the blue-light sensing BLUF domain that controls AppA activity. Journal of Molecular Biology 368, 1223-1230, doi:10.1016/j.jmb.2007.02.087 (2007).

31 Jung, A., Reinstein, J., Domratcheva, T., Shoeman, R. L. & Schlichting, I. Crystal structures of the AppA BLUF domain photoreceptor provide insights into blue light-mediated signal transduction. Journal of Molecular Biology 362, 717-732, doi:10.1016/j.jmb.2006.07.024 (2006).

32 Gauden, M. et al. Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor. Proc. Natl. Acad. Sci. U. S. A. 103, 10895-10900, doi:10.1073/pnas.0600720103 (2006).

33 Stelling, A. L., Ronayne, K. L., Nappa, J., Tonge, P. J. & Meech, S. R. Ultrafast structural dynamics in BLUF domains: Transient infrared spectroscopy of AppA and its mutants. Journal of the American Chemical Society 129, 15556-15564 (2007).

34 Domratcheva, T., Grigorenko, B. L., Schlichting, I. & Nemukhin, A. V. Molecular models predict light-induced glutamine tautomerization in BLUF photoreceptors. Biophysical Journal 94, 3872-3879 (2008).

35 Khrenova, M. G., Nemukhin, A. V. & Domratcheva, T. Photoinduced Electron Transfer Facilitates Tautomerization of the Conserved Signaling Glutamine Side Chain in BLUF Protein Light Sensors. Journal of Physical Chemistry B 117, 2369-2377, doi:10.1021/jp312775x (2013).

36 Collette, F., Renger, T. & Schmidt am Busch, M. Revealing the Functional States in the Active Site of BLUF Photoreceptors from Electrochromic Shift Calculations. The Journal of Physical Chemistry B 118, 11109-11119, doi:10.1021/jp506400y (2014).

30

37 Hsiao, Y.-W., Goetze, J. P. & Thiel, W. The Central Role of Gln63 for the Hydrogen Bonding Network and UV-Visible Spectrum of the AppA BLUF Domain. Journal of Physical Chemistry B 116, 8064-8073, doi:10.1021/jp3028758 (2012).

38 Udvarhelyi, A. & Domratcheva, T. Glutamine Rotamers in BLUF Photoreceptors: A Mechanistic Reappraisal. The Journal of Physical Chemistry B 117, 2888-2897, doi:10.1021/jp400437x (2013).

39 Bonetti, C. et al. Hydrogen Bond Switching among Flavin and Amino Acid Side Chains in the BLUF Photoreceptor Observed by Ultrafast Infrared Spectroscopy. Biophysical Journal 95, 4790-4802, doi:10.1529/biophysj.108.139246 (2008).

40 Lukacs, A. et al. Photoexcitation of the Blue Light Using FAD Photoreceptor AppA Results in Ultrafast Changes to the Protein Matrix. Journal of the American Chemical Society 133, 16893-16900, doi:10.1021/ja2060098 (2011).

41 Lukacs, A. et al. BLUF Domain Function Does Not Require a Metastable Radical Intermediate State. Journal of the American Chemical Society 136, 4605-4615, doi:10.1021/ja4121082 (2014).

42 Greetham, G. M. et al. ULTRA: A Unique Instrument for Time-Resolved Spectroscopy. Applied Spectroscopy 64, 1311-1319 (2011).

43 Towrie, M. et al. Development of a broadband picosecond infrared spectrometer and its incorporation into an existing ultrafast time-resolved resonance Raman, UV/visible, and fluorescence spectroscopic apparatus. Applied Spectroscopy 57, 367-380 (2003).

44 Snellenburg, J. J., Laptenok, S. P., Seger, R., Mullen, K. M. & van Stokkum, I. H. M. Glotaran: A Java-Based Graphical User Interface for the R Package TIMP. Journal of Statistical Software 49, 1-22 (2012).

45 Gauden, M. et al. On the role of aromatic side chains in the photoactivation of BLUF domains. Biochemistry 46, 7405-7415, doi:10.1021/bi7006433 (2007).

46 Haigney, A. et al. Ultrafast Infrared Spectroscopy of an Isotope-Labeled Photoactivatable Flavoprotein. Biochemistry 50, 1321-1328, doi:10.1021/bi101589a (2011).

47 Lukacs, A. et al. Excited State Structure and Dynamics of the Neutral and Anionic Flavin Radical Revealed by Ultrafast Transient Mid-IR to Visible Spectroscopy. Journal of Physical Chemistry B 116, 5810-5818, doi:10.1021/jp2116559 (2012).

48 Brust, R. et al. Ultrafast Structural Dynamics of BlsA, a Photoreceptor from the Pathogenic Bacterium Acinetobacter baumannii. The Journal of Physical Chemistry Letters 5, 220-224, doi:10.1021/jz4023738 (2014).

49 Dragnea, V., Waegele, M., Balascuta, S., Bauer, C. & Dragnea, B. Time-resolved spectroscopic studies of the AppA blue-light receptor BLUF domain from Rhodobacter sphaeroides. Biochemistry 44, 15978-15985 (2005).

50 Hasegawa, K., Masuda, S. & Ono, T. Structural intermediate in the photocycle of a BLUF (sensor of blue light using FAD) protein Slr1694 in a cyanobacterium Synechocystis sp PCC6803. Biochemistry 43, 14979-14986 (2004).

51 Zhao, R.-K. et al. Ultrafast transient mid IR to visible spectroscopy of fully reduced flavins. Physical Chemistry Chemical Physics 13, 17642-17648 (2011).

52 Ishikita, H. Light-induced hydrogen bonding pattern and driving force of electron transfer in AppA BLUF domain photoreceptor. Journal of Biological Chemistry 283, 30618-30623, doi:10.1074/jbc.M803864200 (2008).

53 Marcus, R. A. & Sutin, N. ELECTRON TRANSFERS IN CHEMISTRY AND BIOLOGY. Biochimica Et Biophysica Acta 811, 265-322, doi:10.1016/0304-4173(85)90014-x (1985).

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