Verlag des Forschungszentrums Jülich
JUEL-4210
Majerus, Teresa
FT-IR spectroscopy on blue light photoreceptors employing steady-state and time-resolved techniques
195 S., 2006
The present work contributes to an understanding of photoreactions that occur in
the photoreceptor domains LOV and BLUF. Both domains noncovalently bind avin
as a cofactor and undergo a distinct photocycle upon blue light excitation. Light-induced
UV/Vis and FT-IR difference spectroscopy were employed to characterize the
photocycle. The identification of short-lived intermediates was performed using two
separate approaches: by trapping species at cryogenic temperatures and by using time-resolved
FT-IR spectroscopy with microsecond time resolution at room temperature.
The main results for LOV domains include evidence for the formation of different cys-teinyl
adduct forms at 78 K and at room temperature using steady-state cryogenic
measurements. Moreover, secondary photochemistry takes place upon extended illumi-nation.
The marker band for breakage of the C4 - S bond in the adduct was identified.
The N5-H bending mode in the neutral radical was assigned and the carbonyl vibrations
of the chromophore were characterized. In the FTIR difference spectrum of the cys-teinyl
adduct, the phosphate vibrations of the terminal chromophore and the carbonyl
modes of the Q120 side chain were identified and it was shown that both vibrations
play an important role in the photocycle. By applying time-resolved FT-IR difference
spectroscopy with microsecond time resolution, the “missing link” in the mechanism
between the previously characterized triplet state and the signaling intermediate was
investigated for the first time. These results provide the first experimental evidence
that a radical pair mechanism with an electron transfer as the first step in the formation
of the adduct. This is a significant contribution in understanding the photocycle mech-anism
of LOV domains. In the BLUF domains, it was shown that the adenine moiety
did not in uence molecular changes in the photocycle of AppA-BLUF. Furthermore,
the protonated tyrosine vibrations in the dark state were assigned. Significant progress
in understanding the BLUF-domain signaling mechanism came with time-resolved ex-periments
on the W104F mutant. These data yield information on the existence of the
spectrally different species, as well as the known 10 nm blue-shifted intermediate. A
mechanism governing the signaling process in the BLUF domain was proposed. This
mechanism involves deprotonation of Asp-82 in the photobleached state (O) and the
uptake of a proton by His-85. Additionally, it was revealed that Trp-104 plays a role
as a stabilizer of the signaling state. The identification of intermediates in the BLUF
domain photocycle shed light on the reaction mechanism. A ow- ash FT-IR/ATR
technique was developed and successfully tested on bacteriorhodopsin. In the near fu-ture,
this method could be applied to study wild-type LOV and BLUF domains in a
time-resolved manner.
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