Femtosecond Spectroscopic Observations of Initial Intermediates in the Photocycle of the Photoactive Yellow Protein from Ectothiorhodospira halophila

Abstract

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.

Primary steps of the photoactive yellow protein: Isolated chromophore dynamics and protein directed function

“…First, isomerization time for the isolated chromophore is similar to that observed for the same chromophore in the solution phase (1.3 ps) (D. H. Paik, A. Espagne, M. M. Martin, and A.H.Z., unpublished data). Second, unlike the protein case, the chromophore is free from backbone bonding and can undergo other torsions and rotations, and yet the isomerization time is similar to that of the protein (within a few picoseconds) (7)(8)(9)(10)(11). Third, the coherence, which reflects the involvement of low-frequency motion(s) in isomerization (20), is preserved on going from the isolated molecule to the protein.…”

“…The twisted configuration is in a well with a depth of Ϸ1 eV. § For this intermediate, we measured 52 ps for its decay, but in the protein the formation of the ground-state cis isomer (I 0 , the first intermediate of the PYP photocycle) is within a few picoseconds (8)(9)(10)(11). This large discrepancy suggests that the position of CI on the potential energy surface is dependent on the environment.…”

“…Light absorption of the PYP chromophore leads to the initiation of a complex photocycle involving several intermediates. With a variety of biophysical techniques (2)(3)(4)(5), it is concluded that the first intermediate (I 0 ) in the photocycle is formed within a few picoseconds, with the chromophore changing to the cis configuration, in a trans-to-cis isomerization process (7)(8)(9)(10)(11). The intermediates at longer times have been trapped and identified by x-ray crystallography (12,13).…”

Femtosecond Studies of the Initial Events in the Photocycle of Photoactive Yellow Protein (PYP)

“…This measurement, in agreement with that at 487 nm in ref. [29], offers a spectral window on the bleach recovery kinetics of the So states. …”

“…[251 With this kinetic scheme, the measured time constants, and the constraint that the cisltrans quantum yield must be in agreement with that reported in ref. [29], the 400 nm probe data could be fitted by adjusting the inhomogeneous distribution in the Sl(trans) states, the kinetic time constants, and the absorption coefficients of the states involved. The best fit was achieved with the inhomogeneous distribution of the Sl(trans) (and hence also the So (trans) ground state) populations of 0.5 each and a quantum yield of So(cis) of 0.6.…”