Femtosecond time-resolved spectroscopy of the primary photochemistry of phytochrome

Time-resolved absorption spectra of P_r phytochrome were obtained using a regeneratively amplified femtosecond titanium : sapphire laser system. The early time transient absorption spectra are comprised of prompt P_r photobleaching, stimulated emission, and excited-state absorption features that decay with a 24 ps time constant that matches the ground state appearance time of the primary photoproduct. Based on the 5 ns radiative lifetime calculated from the absorption and spontaneous emission spectra and the fluorescence quantum yield of 5.5 (± 0.5) × 10⁻³, we calculate an excited-state lifetime of 28 ps that agrees well with the directly determined lifetime. The transient absorption spectra are consistent with a primary photochemical reaction quantum yield of 0.15, and the absorption spectrum of the primary photoproduct closely resembles that of the low-temperature trapped intermediate, lumi-R. We conclude that the primary photoisomerization, which is believed to be a Z,syn → E,syn isomerization of the C₁₅=C₁₆ chromophore bond, occurs in 24 ps. © 1997 John Wiley & Sons, Inc. Biospectroscopy 3: 421–433, 1997