Femtosecond spectroscopic observations of initial intermediates in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila

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.