Retinal photoisomerization dynamics are studied at both
room temperature and 20 K in wild-type bacteriorhodopsin using femtosecond pulses. We were able to resolve
the decay at 20 K into two components
with the dominant component having a similar lifetime to that observed
at room temperature. This strongly
suggests that the retinal lifetime at physiological temperature is
barrierless. The minor, low-temperature
long-lived component is discussed in terms of previous results obtained
for fluorescence and transient absorption
with lower time resolution, and the origin of this component is
discussed in terms of low-temperature glass
heterogeneity.
Photochemical hole-burning spectroscopy was used to study the excited-state electronic structure of the 4-hydroxycinnamyl chromophore in photoactive yellow protein (PYP). This system is known to undergo a trans-to-cis isomerization process on a femtosecond-to-picosecond time scale, similar to membrane-bound rhodopsins, and is characterized by a broad featureless absorbance at 446 nm. Resolved vibronic structure was observed for the hole-burned spectra obtained when PYP in phosphate buffer at pH 7 was frozen at low temperature and irradiated with narrow bandwidth laser light at 431 nm. The approximate homogeneous width of 752 cm-1 could be calculated from the deconvolution of the hole-burned spectra leading to an estimated dephasing time of approximately 14 fs for the PYP excited-state structure. The resolved vibronic structure also enabled us to obtain an estimated change in the C=C stretching frequency, from 1663 cm-1 in the ground state to approximately 1429 cm-1 upon photoexcitation. The results obtained allowed us to speculate about the excited-state structure of PYP. We discuss the data for PYP in relation to the excited-state model proposed for the photosynthetic membrane protein bacteriorhodopsin, and use it to explain the primary event in the function of photoactive biological protein systems. Photoexcitation was also carried out at 475 nm. The vibronic structure obtained was quite different both in terms of the frequencies and Franck-Condon envelope. The origin of this spectrum was tentatively assigned.
The frequency difference between the symmetric and antisymmetric
stretching vibration of PO2
- in
phosphatidylglycerol phospate (PGP) is used to differentiate between
monodentate and bidentate binding of
these groups to metal cations in the membrane of bacteriorhodopsin (bR)
and phosphatidylglycerol phospate.
The binding of Ca2+ to PGP is found to have a
frequency difference corresponding to monodentate binding.
The symmetric and antisymmetric PO2
-
bands in bR show similar frequency shifts upon Ca2+
binding, which
is independent of pH. This suggests that Ca2+ has a
monodentate type binding with the PO2
- in bR.
In
contrast, the PO2
- symmetric and
antisymmetric frequencies of PGP complexes with trivalent
lanthanide
cations with higher charge density (Ho3+ and
Dy3+) are observed to have smaller separations and to
increase
their separation with increasing pH toward the value observed for
Ca2+ binding. Lanthanide cations
(Ho3+,
Dy3+, Eu3+, Nd3+, and
La3+) binding in bR at pH 4 show small frequency
separations that are observed to
have similar frequency shifts with pH, the magnitude of which is
dependent on the cation. It is proposed that
at low pH the lanthanide cations with higher charge density have
bidentate binding to bR, while at high pH,
complexation with the OH- competes with one of the
oxygens of the PO2
- for the binding of the
lanthanide
ion thus changing the bidentate to monodentate type
binding.
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