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: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al. 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 (I0) exhibits maximum absorption at approximately 510 nm and appears in </=3 ps after 452 nm excitation (5 ps pulse width) of PYP. Kinetic analysis shows that I0 decays with a 220 +/- 20 ps lifetime, forming another intermediate (Idouble dagger0) that has a similar difference wavelength maximum, but with lower absorptivity. Idouble dagger0 decays with a 3 +/- 0.15 ns time constant to form I1. 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.
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Department of Chemistry and Optical Science Center, University of Arizona, Tucson, Arizona 85721 USA.
Devanathan
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Meyer
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Cusanovich
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Tollin
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Atkinson
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D055592:Biophysical Phenomena; D001703:Biophysics; D002841:Chromatiaceae / Q000737:chemistry* / Q000528:radiation effects; D007700:Kinetics; D008027:Light; D010777:Photochemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000528:radiation effects; D013053:Spectrophotometry
D011994:Recombinant Proteins