New photocycle intermediates in the photoactive yellow protein from Ectothiorhodospira halophila:: Picosecond transient absorption spectroscopy

Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I-1, which forms in <10 ns and decays with a 200-mu s lifetime to I-2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et ai. 1997. Biochemistry. 36:8-14). Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously. The first new intermediate (I-0) exhibits maximum absorption at similar to 510 nm and appears in less than or equal to 3 ps after 452 nm excitation (5 ps pulse width) of PYP. Kinetic analysis shows that I-0 decays with a 220 +/- 20 ps lifetime, forming another intermediate (I double dagger(0)) that has a similar difference wavelength maximum, but with lower absorptivity. I double dagger(0) decays with a 3 +/- 0.15 ns time constant to form I-1. Stimulated emission from an excited electronic state of PYP is observed both within the 4-6-ps cross-correlation times used in this work, and with a 16-ps delay for all probe wavelengths throughout the 426-525-nm region studied. These transient absorption and emission data provide a more detailed understanding of the mechanistic dynamics occurring during the PYP photocycle.