Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al. 1997. Biochemistry. 36:8-14). Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously. The first new intermediate (I0) exhibits maximum absorption at approximately 510 nm and appears in =3 ps after 452 nm excitation (5 ps pulse width) of PYP. Kinetic analysis shows that I0 decays with a 220 +/- 20 ps lifetime, forming another intermediate (Idouble dagger0) that has a similar difference wavelength maximum, but with lower absorptivity. Idouble dagger0 decays with a 3 +/- 0.15 ns time constant to form I1. Stimulated emission from an excited electronic state of PYP is observed both within the 4-6-ps cross-correlation times used in this work, and with a 16-ps delay for all probe wavelengths throughout the 426-525-nm region studied. These transient absorption and emission data provide a more detailed understanding of the mechanistic dynamics occurring during the PYP photocycle.
Femtosecond time-resolved absorbance measurements were used to probe the subpicosecond primary events of the photoactive yellow protein (PYP), a 14-kD soluble photoreceptor from Ectothiorhodospira halophila. Previous picosecond absorption studies from our laboratory have revealed the presence of two new early photochemical intermediates in the PYP photocycle, I(0), which appears in =3 ps, and I(0)(double dagger), which is formed in 220 ps, as well as stimulated emission from the PYP excited state. In the present study, kinetic measurements at two excitation wavelengths (395 nm and 460 nm) on either side of the PYP absorption maximum (446 nm) were undertaken using 100-fs pump and probe pulses. Global analysis over a range of probe wavelengths yielded time constants of 1.9 ps for the photochemical formation of the I(0) intermediate via the PYP excited state, and 3.4 ps for the repopulation of the ground state from the excited state. In addition to these pathways, 395 nm excitation also initiated an alternative route for PYP excitation and photochemistry, presumably involving a different excited electronic state of the chromophore. No photochemical intermediates formed before I(0) were observed. Based on these data, a quantum yield of 0.5-0.6 for I(0) formation was determined. The structural and mechanistic aspects of these results are discussed.
The vibrational spectrum (800-1700 cm -1 region) of the J-625 intermediate, formed within 200-500 fs (3.5 ps decay time to K-590) in the room-temperature bacteriorhodopsin (BR) photocycle, is measured via picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). An examination of the excitation conditions and BR photocycle kinetics, as well as the vibrational CARS data, convincingly demonstrates that these PTR/CARS spectra can be quantitatively analyzed in terms of primarily BR-570 and J-625 by using third-order nonlinear susceptibility (χ (3) ) relationships. The resultant background-free (Lorentzian line shapes) CARS spectrum contains 24 distinct vibrational features which provide the most complete structural characterization of J-625 yet reported. Comparisons of the J-625 vibrational spectrum with those of groundstate BR-570 and the K-590 intermediate show that J-625 maintains some structural similarities with BR-570 while it has a significantly different structure than that of K-590. Specifically, J-625 has (i) an all-trans retinal configuration, (ii) increased electron density in the CdC stretching modes as manifested by increased CdC stretching frequencies relative to those in both BR-570 and K-590, (iii) significant delocalized hydrogen out-of-plane motion not observed in any other BR species, (iv) decreased C-CH 3 in-plane wagging motion, and (v) a Schiff-base bonding environment similar to that of BR-570 and distinctively different from that in K-590. Comparisons between the PTR/CARS spectra of J-625 and T5.12, an intermediate found in the photoreaction of the artificial BR pigment, BR5.12, containing a five-membered ring spanning the C 12 -C 13 dC 14 bonds (thereby blocking C 13 dC 14 isomerization), support the conclusion that the J-625 structure reflects the reaction coordinates in the BR photocycle that precede C 13 dC 14 isomerization. Since these PTR/ CARS data show J-625 to have an all-trans retinal, C 13 dC 14 isomerization cannot be the primary reaction coordinate described in numerous models for the BR photocycle. The all-trans to 13-cis isomerization must occur as J-625 transforms into K-590, and other changes in the retinal structural and/or retinal-protein interactions must comprise the primary reaction coordinates that precede C 13 dC 14 isomerization. These results require that significant changes in the mechanistic model describing the room-temperature BR photocycle be considered.
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