Probing the Primary Event in the Photocycle of Photoactive Yellow Protein Using Photochemical Hole-burning Technique¶

Abstract

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.

Deuterium Isotope Effects in the Photocycle Transitions of the Photoactive Yellow Protein

“…In an attempt to simulate a ground state spectrum we found that the ground state spectrum can be simulated very well by two skewed Gaussians (Fraser and Suzuki, 1969;Sevilla et al, 1989) above 385 nm. The maxima of these two skewed Gaussians were selected to be at 425 and 452.4 nm, values that have been observed in a low temperature study (Masciangioli et al, 2000). We determined an appropriate calculated ground state spectrum via a global analysis of five time-gated difference spectra that did not contain a pR signal, using the Origin 6.0 program.…”

Contrasting the Excited-State Dynamics of the Photoactive Yellow Protein Chromophore: Protein versus Solvent Environments

“…The sensitivity of the excited-state dynamics to the arrangement of the pigment binding pocket has also been demonstrated experimentally by Mataga et al (2000a) by investigating different mutants of PYP. Inhomogeneous broadening in PYP has been established by earlier holeburning experiments (Masciangioli et al, 2000), whereas the existence of the energy barrier for the isomerization has been suggested by low-temperature fluorescence excitation measurements (Hoff et al, 1992).…”

Quantum Mechanical/Molecular Mechanical Studies on Spectral Tuning Mechanisms of Visual Pigments and Other Photoactive Proteins†

“…PYP has a λ max of 446 nm, while the bare chromophore (pCA) absorbs light at 460 nm in the gas phase (7,8). PYP is a very flexible protein and the hole‐burning experiment of PYP at 10 K indicates an inhomogeneous ground state (62). In agreement with this experimental result (62), CASPT2/AMBER excitation energy calculations (QM region: pCA and carboxyl group of Glu46) on 10 snapshots of a short MD trajectory find λ max of 455 (4 snapshots), 415 (one snapshot) and 390 (5 snapshots) nm (63,64).…”