S. Devanathan et al., Femtosecond spectroscopic observations of initial intermediates in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila, BIOPHYS J, 77(2), 1999, pp. 1017-1023
Femtosecond time-resolved absorbance measurements were used to probe the su
bpicosecond primary events of the photoactive yellow protein (PYP), a 14-kD
soluble photoreceptor from Ectothiorhodospira halophila. Previous picoseco
nd absorption studies from our laboratory have revealed the presence of two
new early photochemical intermediates in the PYP photocycle, I-0, which ap
pears in less than or equal to 3 ps, and I-0(double dagger), which is forme
d 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 u
ndertaken 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 fo
r the repopulation of the ground state from the excited state. In addition
to these pathways, 395 nm excitation also initiated an alternative route fo
r PYP excitation and photochemistry, presumably involving a different excit
ed electronic state of the chromophore. No photochemical intermediates form
ed 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.