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

Savitha Devanathan, Andrew Pacheco, Laszlo Ujj, Michael Cusanovich, Gordon Tollin, Su Lin, Neal Woodbury

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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(o), which appears in ≤3 ps, and I(o)/((+)), 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(o) 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(o) were observed. Based on these data, a quantum yield of 0.5-0.6 for I(o) formation was determined. The structural and mechanistic aspects of these results are discussed.

Original languageEnglish (US)
Pages (from-to)1017-1023
Number of pages7
JournalBiophysical journal
Issue number2
StatePublished - Aug 1999


ASJC Scopus subject areas

  • Biophysics

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