Transient Absorption Spectroscopy of Slr1694.
The Slr1694 protein was excited at 400 nm, and the time-resolved
absorbance changes were monitored over a wavelength range from 420 to
720 nm. The data were globally analyzed in terms of a kinetic scheme
with sequentially interconverting species, where each species is
characterized by an evolution-associated difference spectrum (EADS).
Five components were required for an adequate description of the
time-resolved data, with lifetimes of 0.9, 7, 22, and 123 ps and a
long-lived component. The resulting EADS are presented in Fig. 2A, and selected kinetic traces at 483, 550, 610, and 710 nm are shown in Fig. 3 as the open circles. The first EADS (black curve) in Fig. 2A can be assigned to the initially created “hot” S1
singlet excited state of the FAD chromophore, which shows a ground
state bleach at wavelengths 425–475 nm, a stimulated emission band near
550 nm, and excited-state absorption at wavelengths >600 nm. This
EADS evolves in 0.9 ps to the next EADS (red line), which has a
lifetime of 7 ps. The 0.9-ps evolution is attributed to a vibrational
cooling process of the FAD excited state (18)
and involves a small blue-shift of the stimulated emission band and an
increase of excited-state absorption of ≈510 nm, whereas the
ground-state bleach remains the same. The EADS with a 7-ps lifetime
thus corresponds to the vibrationally relaxed singlet excited state of
FAD (hereafter denoted FAD*) and evolves to the next EADS (green line),
which has a lifetime of 22 ps. This component shows a decrease of the
stimulated emission band at 550 nm and the absorption at wavelengths
>600 nm, indicating decay of FAD*. Additionally, an increase of
absorption of ≈510 nm and the appearance of a distinct absorption near
600 nm is observed, whereas the ground state bleach remains the same.
Thus, the 22-ps EADS can be assigned to a remaining fraction of FAD*
but with a contribution of another molecular species to be determined
below. The evolution in 22 ps to the next EADS (blue line), which has a
lifetime of 123 ps, corresponds to a further loss of the stimulated
emission band and partial loss of the ground-state bleach. The induced
absorption bands at 510 and 600 nm are still present, and,
additionally, a shoulder near 490 nm is formed.
In the fifth, nondecaying EADS (cyan line), the ground-state bleach
has largely disappeared, and a narrow absorption feature near 483 nm
has appeared. The nondecaying EADS strongly resembles the absorption of
the long-lived signaling state in Slr1694 (17).
We conclude that in Slr1694, the red-shifted signaling state is formed
in 123 ps, consistent with previous results on the AppA BLUF domain
where the long-lived product is formed on the sub-nanosecond timescale (18).
Similar to AppA, FAD* in Slr1694 decays multiexponentially, albeit with
substantially shorter lifetimes as judged by the decay of the
stimulated emission band and the kinetic trace taken at 550 nm. Most
significantly, the appearance of photocycle intermediates is observed
in Slr1694, which contrasts with the situation in the AppA BLUF domain (18).
Effects of H/D Exchange.
To investigate whether the observed reaction intermediates are
kinetically coupled by means of PT, ultrafast experiments were
performed with deuterated Slr1694. The EADS that resulted from the
sequential analysis were similar to those obtained with Slr1694 in H2O and are presented in Fig. 2B. Kinetic traces are shown in Fig. 3 as the filled circles. The lifetimes of 2.5, 11, 52, and 210 ps were consistently longer than for Slr1694 in H2O,
indicating a pronounced kinetic isotope effect (KIE) upon H/D exchange
of Slr1694. The 210-ps EADS (blue line) is somewhat different from the
corresponding 123-ps EADS of Slr1694 in H2O because the
contribution by FAD* seems to be smaller in the former. Interestingly,
the shape of the 210-ps EADS in the spectral region from 500 to 700 nm
strongly resembles that of a neutral flavin semiquinone FADH• radical (19),
indicating the involvement of flavin radical species in the Slr1694
photoreaction, a point that will be substantiated below. The yields of
signaling-state formation for Slr1694 in H2O and in D2O
are identical, as evident from the amplitude of the 483-nm absorption
(cyan line) relative to that of the initial ground-state bleach signal
of the first EADS (black line) in each preparation. The relative
amplitude of the long-lived product absorption is ≈1.6 times higher in
Slr1694 as compared with AppA (18).
For AppA, an absolute quantum yield of 24% was determined, implying
that the quantum yield of signaling-state formation in Slr1694 amounts
represents selected kinetic traces together with the fitting result of
the target analysis described below (solid line), taken at 483, 550,
610, and 710 nm, respectively. At 550 nm, where stimulated emission
from FAD* has a main contribution, the initial decay phases of Slr1694
in H2O and D2O are almost overlaid, suggesting that no or little KIE applies to the FAD* lifetime (Fig. 3B).
This phenomenon is demonstrated even more clearly in the trace at 710
nm, where FAD* has a pronounced absorption but where most other flavin
species like semiquinone radicals do not absorb: the traces measured in
H2O and D2O are completely overlaid (Fig. 3D).
These observations are consistent with previous results on AppA, which
showed no H/D exchange effect on the excited-state lifetimes (20).
FAD* has an isosbestic point at 610 nm, giving a clean view on the
kinetic behavior of the intermediate species. The trace shows a slower
rise and slower decay in D2O, revealing the existence of two pronounced KIEs (Fig. 3C). In particular, in Slr1694 in D2O, the intermediate decay takes approximately twice as long as in H2O and occurs in ≈200 ps. The KIE can also be observed in the 483-nm trace where the rise in D2O is slower than in H2O (Fig. 3A).
Thus, the decay of the intermediate state is kinetically coupled to the
rise of the long-lived product, demonstrating that the intermediate is
on the reaction pathway of product formation.
Target Analysis: Identification of Intermediate Reaction Dynamics.
The femtosecond transient absorption data on Slr1694 show that
essentially at all delay times, a mixture of FAD*, reaction
intermediate(s), or long-lived product states make up the transient
spectra. To disentangle the contributions by the various molecular
species, we performed a target analysis of time-resolved data of
hydrated and deuterated Slr1694 samples, wherein the data are described
in terms of a kinetic scheme, thereby identifying their spectral
signatures and estimating their lifetimes and their connectivity. A
similar approach was taken recently to dissect the PT dynamics in GFP (21).
The kinetic scheme used for the target analysis, the transient
concentrations of the molecular species involved, and the residuals of
the fitting procedure are provided in Figs. 6 and 7 and Supporting Text,
which are published as supporting information on the PNAS web site,
along with a detailed description of the applied assumptions and
premises. In short, four lifetimes are assumed for the decay of FAD*,
which stem from inhomogeneity in the ground-state population as shown
for the AppA BLUF domain (14, 18). FAD* evolves to a first intermediate Q1, which in turn evolves to a second intermediate Q2. From Q2 the long-lived photoproduct SlrRED is formed. To account for the quantum yield for SlrRED
formation of ≈40%, a loss process must be included in the kinetic
scheme. Because the ground-state bleach in the sequential analysis does
not diminish after the first decay phase of FAD* (see Fig. 2), the loss process is introduced on the transition from Q1 to Q2.
The rate constants, fractions, and quantum yields are summarized in
Table 1, which is published as supporting information on the PNAS web
Fig. 4 shows the species-associated difference spectra (SADS) of FAD*, Q1, Q2, and SlrRED. As expected, the SADS of FAD* has the typical shape of the singlet-excited state as determined for FAD in solution (22) and in AppA (18) and as they follow from the sequential analysis in Fig. 2. The SADS of SlrRED is virtually identical to the red-shifted intermediate observed in steady state, with a main absorption peak at 483 nm (17). The SADS of Q2
has a broad absorption in the 480- to 650-nm region with peaks at 510,
566, and 610 nm. This SADS strongly resembles the spectrum of a neutral
semiquinone flavin radical, FADH• (19). It is this species that is mainly responsible for the pronounced absorption around ≈510 and ≈600 nm in the 123-ps (H2O) and 210-ps (D2O) EADS in the sequential analysis, shown in Fig. 2.
The SADS of Q1 shows an absorption at ≈510 nm and a broad
symmetric band at ≈590 nm. The symmetric structure of the latter
suggests that it corresponds to a flavin charge-transfer (CT) band
rather than of a neutral semiquinone (23). The shape and amplitude of the Q1 SADS is generally similar to that of anionic semiquinone FAD•− in a CT interaction as observed in d-amino
acid oxidase. The latter has a narrow absorption peak with a maximum at
≈500 nm and a broad CT band with a maximum at ≈700 nm (23, 24).
Given that the absorption maximum of flavin CT bands may vary widely
from one flavoprotein to another and depend on specific interactions
between flavin and protein, we identify the intermediate species Q1 as a CT/anionic semiquinone FAD•− species.
Fig. 4 Lower
shows a photocycle scheme for the photoactivation of Slr1694, based on
the results from the target analysis. The initial light-driven reaction
from FAD* involves ET from the protein environment to result in FAD•−.
This event takes place multiexponentially and is insensitive to H/D
exchange, with a dominant time constant of 7 ps. The resulting anionic
flavin radical FAD•− is rapidly protonated in 6 ps to result in the neutral semiquinone radical FADH•. This reaction slows down 3-fold in D2O,
which is consistent with a putative PT event from the protein
environment to FAD. Note that as a result of rate-limiting formation
times, the FAD•− radical only accumulates transiently
between 5 and 15 ps. This effect can be seen from the transient
concentration profiles of FAD*, Q1, Q2, and SlrRED, shown in Fig. 6. Finally, the signaling state SlrRED is formed in 65 ps from the FADH• neutral radical. This reaction is 2.4 times slower in D2O, which is reasonable because SlrRED
corresponds to an oxidized FAD. The deuteration effect on the anionic
and neutral radical lifetimes, and on the rise time of SlrRED, proves that the radicals are in fact intermediates on the pathway to signaling-state formation. Formation of FAD•−
occurs stoichiometrically from FAD*, but ≈50% recombines to the ground
state before its protonation results in formation of FADH•.