8 research outputs found
Ultrafast Proton Shuttling in <i>Psammocora</i> Cyan Fluorescent Protein
Cyan, green, yellow, and red fluorescent
proteins (FPs) homologous
to green fluorescent protein (GFP) are used extensively as model systems
to study fundamental processes in photobiology, such as the capture
of light energy by protein-embedded chromophores, color tuning by
the protein matrix, energy conversion by FoĢrster resonance
energy transfer (FRET), and excited-state proton transfer (ESPT) reactions.
Recently, a novel cyan fluorescent protein (CFP) termed psamFP488
was isolated from the genus <i>Psammocora</i> of reef building
corals. Within the cyan color class, psamFP488 is unusual because
it exhibits a significantly extended Stokes shift. Here, we applied
ultrafast transient absorption and pumpādumpāprobe spectroscopy
to investigate the mechanistic basis of psamFP488 fluorescence, complemented
with fluorescence quantum yield and dynamic light scattering measurements.
Transient absorption spectroscopy indicated that, upon excitation
at 410 nm, the stimulated cyan emission rises in 170 fs. With pumpādumpāprobe
spectroscopy, we observe a very short-lived (110 fs) ground-state
intermediate that we assign to the deprotonated, anionic chromophore.
In addition, a minor fraction (14%) decays with 3.5 ps to the ground
state. Structural analysis of homologous proteins indicates that Glu-167
is likely positioned in sufficiently close vicinity to the chromophore
to act as a proton acceptor. Our findings support a model where unusually
fast ESPT from the neutral chromophore to Glu-167 with a time constant
of 170 fs and resulting emission from the anionic chromophore forms
the basis of the large psamFP488 Stokes shift. When dumped to the
ground state, the proton on neutral Glu is very rapidly shuttled back
to the anionic chromophore in 110 fs. Proton shuttling in excited
and ground states is a factor of 20ā4000 faster than in GFP,
which probably results from a favorable hydrogen-bonding geometry
between the chromophore phenolic oxygen and the glutamate acceptor,
possibly involving a short hydrogen bond. At any time in the reaction,
the proton is localized on either the chromophore or Glu-167, which
implies that most likely no low-barrier hydrogen bond exists between
these molecular groups. This work supports the notion that proton
transfer in biological systems, be it in an electronic excited or
ground state, can be an intrinsically fast process that occurs on
a 100 fs time scale. PsamFP488 represents an attractive model system
that poses an ultrafast proton transfer regime in discrete steps.
It constitutes a valuable model system in addition to wild type GFP,
where proton transfer is relatively slow, and the S65T/H148D GFP mutant,
where the effects of low-barrier hydrogen bonds dominate
Molecular Origin of Photoprotection in Cyanobacteria Probed by Watermarked Femtosecond Stimulated Raman Spectroscopy
Photoprotection
is fundamental in photosynthesis to avoid oxidative
photodamage upon excess light exposure. Excited chlorophylls (Chl)
are quenched by carotenoids, but the precise molecular origin remains
controversial. The cyanobacterial HliC protein belongs to the Hlip
family ancestral to plant light-harvesting complexes, and binds Chl <i>a</i> and Ī²-carotene in 2:1 ratio. We analyzed HliC by
watermarked femtosecond stimulated Raman spectroscopy to follow the
time evolution of its vibrational modes. We observed a 2 ps rise of
the Cī»C stretch band of the 2A<sub>g</sub><sup>ā</sup> (S<sub>1</sub>) state of Ī²-carotene upon Chl <i>a</i> excitation, demonstrating energy transfer quenching and fast excess-energy
dissipation. We detected two distinct Ī²-carotene conformers
by the Cī»C stretch frequency of the 2A<sub>g</sub><sup>ā</sup> (S<sub>1</sub>) state, but only the Ī²-carotene whose 2A<sub>g</sub><sup>ā</sup> energy level is significantly lowered
and has a lower Cī»C stretch frequency is involved in quenching.
It implies that the low carotenoid S<sub>1</sub> energy that results
from specific pigmentāprotein or pigmentāpigment interactions
is the key property for creating a dissipative energy channel. We
conclude that watermarked femtosecond stimulated Raman spectroscopy
constitutes a promising experimental method to assess energy transfer
and quenching mechanisms in oxygenic photosynthesis
Unfolding of the CāTerminal JĪ± Helix in the LOV2 Photoreceptor Domain Observed by Time-Resolved Vibrational Spectroscopy
Light-triggered
reactions of biological photoreceptors have gained
immense attention for their role as molecular switches in their native
organisms and for optogenetic application. The light, oxygen, and
voltage 2 (LOV2) sensing domain of plant phototropin binds a C-terminal
JĪ± helix that is docked on a Ī²-sheet and unfolds upon
light absorption by the flavin mononucleotide (FMN) chromophore. In
this work, the signal transduction pathway of LOV2 from Avena sativa was investigated using time-resolved
infrared spectroscopy from picoseconds to microseconds. In D<sub>2</sub>O buffer, FMN singlet-to-triplet conversion occurs in 2 ns and formation
of the covalent cysteinyl-FMN adduct in 10 Ī¼s. We observe a
two-step unfolding of the JĪ± helix: The first phase occurs concomitantly
with Cys-FMN covalent adduct formation in 10 Ī¼s, along with
hydrogen-bond rupture of the FMN C4ī»O with Gln-513, motion
of the Ī²-sheet, and an additional helical element. The second
phase occurs in approximately 240 Ī¼s. The final spectrum at
500 Ī¼s is essentially identical to the steady-state light-minus-dark
Fourier transform infrared spectrum, indicating that JĪ± helix
unfolding is complete on that time scale
Hydrogen Bond Switching among Flavin and Amino Acids Determines the Nature of Proton-Coupled Electron Transfer in BLUF Photoreceptors
BLUF domains are flavin-binding photoreceptors that can
be reversibly
switched from a dark-adapted state to a light-adapted state. Proton-coupled
electron transfer (PCET) from a conserved tyrosine to the flavin that
results in a neutral flavin semiquinone/tyrosyl radical pair constitutes
the photoactivation mechanism of BLUF domains. Whereas in the dark-adapted
state PCET occurs in a sequential fashion where electron transfer
precedes proton transfer, in the light-adapted state the same radical
pair is formed by a concerted mechanism. We propose that the altered
nature of the PCET process results from a hydrogen bond switch between
the flavin and its surrounding amino acids that preconfigures the
system for proton transfer. Hence, BLUF domains represent an attractive
biological model system to investigate and understand PCET in great
detail
Photoadduct Formation from the FMN Singlet Excited State in the LOV2 Domain of Chlamydomonas reinhardtii Phototropin
The
two light, oxygen, and voltage domains of phototropin are blue-light
photoreceptor domains that control various functions in plants and
green algae. The key step of the light-driven reaction is the formation
of a photoadduct between its FMN chromophore and a conserved cysteine,
where the canonical reaction proceeds through the FMN triplet state.
Here, complete photoreaction mapping of CrLOV2 from Chlamydomonas reinhardtii phototropin and AsLOV2
from Avena sativa phototropin-1 was
realized by ultrafast broadband spectroscopy from femtoseconds to
microseconds. We demonstrate that in CrLOV2, a direct photoadduct
formation channel originates from the initially excited singlet state,
in addition to the canonical reaction through the triplet state. This
direct photoadduct reaction is coupled by a proton or hydrogen transfer
process, as indicated by a significant kinetic isotope effect of 1.4
on the fluorescence lifetime. Kinetic model analyses showed that 38%
of the photoadducts are generated from the singlet excited state
Unraveling the Carrier Dynamics of BiVO<sub>4</sub>: A Femtosecond to Microsecond Transient Absorption Study
Bismuth vanadate (BiVO<sub>4</sub>) is a promising semiconductor material for photoelectrochemical
water splitting showing good visible light absorption and a high photochemical
stability. To improve the performance of BiVO<sub>4</sub>, it is of
key importance to understand its photophysics upon light absorption.
Here we study the carrier dynamics of BiVO<sub>4</sub> prepared by
the spray pyrolysis method using broadband transient absorption spectroscopy
(TAS), in thin films as well as in a photoelectrochemical (PEC) cell
under water-splitting conditions. The use of a dual-laser setup consisting
of electronically synchronized Ti:sapphire amplifiers enable us to
measure the femtosecond to microsecond time scales in a single experiment.
On the basis of this data, we propose a model of carrier dynamics
that includes relaxation and trapping rates for electrons and holes.
Hole trapping occurs in multiple phases, with the majority of the
photogenerated holes being trapped with a time constant of 5 ps and
a small fraction of this hole trapping taking place within the instrument
response of 120 fs. The induced absorption band that represents the
trapped holes is modulated by an oscillation of 63 cm<sup>ā1</sup>, which is assigned to the coupling of holes to a phonon mode. We
find electrons to undergo a relaxation with a time constant of 40
ps, followed by deeper trapping on the 2.5 ns time scale. On time
scales longer than 10 ns, trap-limited recombination that follows
a power law is found, spanning time scales up to microseconds. Finally,
we observe no spectral or kinetic differences by applying a bias voltage
to the PEC cell, indicating that the effect of a voltage and the charge
transfer processes between BiVO<sub>4</sub> and the electrolyte occurs
on longer time scales. Our results therefore provide new insights
into the carrier dynamics of BiVO<sub>4</sub> and further expand the
application window of TAS as an analytical tool for photoanode materials
Flavin Adenine Dinucleotide Chromophore Charge Controls the Conformation of Cyclobutane Pyrimidine Dimer Photolyase Ī±āHelices
Observations of light-receptive enzyme
complexes are usually complicated
by simultaneous overlapping signals from the chromophore, apoprotein,
and substrate, so that only the initial, ultrafast, photonāchromophore
reaction and the final, slow, protein conformational change provide
separate, nonoverlapping signals. Each provides its own advantages,
whereas sometimes the overlapping signals from the intervening time
scales still cannot be fully deconvoluted. We overcome the problem
by using a novel method to selectively isotope-label the apoprotein
but not the flavin adenine dinucleotide (FAD) cofactor. This allowed
the Fourier transform infrared (FTIR) signals to be separated from
the apoprotein, FAD cofactor, and DNA substrate. Consequently, a comprehensive
structureāfunction study by FTIR spectroscopy of the <i>Escherichia coli</i> cyclobutane pyrimidine dimer photolyase
(CPD-PHR) DNA repair enzyme was possible. FTIR signals could be identified
and assigned upon FAD photoactivation and DNA repair, which revealed
protein dynamics for both processes beyond simple one-electron reduction
and ejection, respectively. The FTIR data suggest that the synergistic
cofactorāprotein partnership in CPD-PHR linked to changes in
the shape of FAD upon one-electron reduction may be coordinated with
conformational changes in the apoprotein, allowing it to fit the DNA
substrate. Activation of the CPD-PHR chromophore primes the apoprotein
for subsequent DNA repair, suggesting that CPD-PHR is not simply an
electron-ejecting structure. When FAD is activated, changes in its
structure may trigger coordinated conformational changes in the apoprotein
and thymine carbonyl of the substrate, highlighting the role of Glu275.
In contrast, during DNA repair and release processes, primary conformational
changes occur in the enzyme and DNA substrate, with little contribution
from the FAD cofactor and surrounding amino acid residues
Spectroscopic Analysis of a Biomimetic Model of Tyr<sub>Z</sub> Function in PSII
Using
natural photosynthesis as a model, bio-inspired constructs
for fuel generation from sunlight are being developed. Here we report
the synthesis and time-resolved spectroscopic analysis of a molecular
triad in which a porphyrin electron donor is covalently linked to
both a cyanoporphyrin electron acceptor and a benzimidazoleāphenol
model for the Tyr<sub>Z</sub>-D<sub>1</sub>His190 pair of PSII. A
dual-laser setup enabled us to record the ultrafast kinetics and long-living
species in a single experiment. From this data, the photophysical
relaxation pathways were elucidated for the triad and reference compounds.
For the triad, quenching of the cyanoporphyrin singlet excited state
lifetime was interpreted as photoinduced electron transfer from the
porphyrin to the excited cyanoporphyrin. In contrast to a previous
study of a related molecule, we were unable to observe subsequent
formation of a long-lived charge separated state involving the benzimidazoleāphenol
moiety. The lack of detection of a long-lived charge separated state
is attributed to a change in energetic landscape for charge separation/recombination
due to small differences in structure and solvation of the new triad