Primary Photoreaction of Photoactive Yellow Protein Studied by Subpicosecond-Nanosecond Spectroscopy † Yasushi Imamoto,* ,‡ Mikio Kataoka, ‡ Fumio Tokunaga, § Tsuyoshi Asahi, | and Hiroshi Masuhara | Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan, Department of Earth and Space Science, Graduate School of Science, Osaka UniVersity, Toyonaka, Osaka 560-0043, Japan, and Department of Applied Physics, Graduate School of Engineering, Osaka UniVersity, Suita, Osaka 565-0871, Japan ReceiVed October 20, 2000; ReVised Manuscript ReceiVed March 7, 2001 ABSTRACT: The primary photochemical event of photoactive yellow protein (PYP) was studied by laser flash photolysis experiments on a subpicosecond-nanosecond time scale. PYP was excited by a 390-nm pulse, and the transient difference absorption spectra were recorded by a multichannel spectrometer for a more reliable spectral analysis than previously possible. Just after excitation, an absorbance decrease due to the stimulated emission at 500 nm and photoconversion of PYP at 450 nm were observed. The stimulated emission gradually shifted to 520 nm and was retained up to 4 ps. Then, the formation of a red-shifted intermediate with a broad absorption spectrum was observed from 20 ps to 1 ns. Another red-shifted intermediate with a narrow absorption spectrum was formed after 2 ns and was stable for at least 5 ns. The latter is therefore believed to correspond to I1 (PYP L ), which has been detected on a nanosecond time scale or trapped at -80 °C. Singular value decomposition analysis demonstrated that the spectral shifts observed from 0.5 ps to 5 ns could be explained by two-component decay of excited state(s) and conversion from PYP B to PYP L . The amount of PYP L at 5 ns was less than that of photoconverted PYP, suggesting the formation of another intermediate, PYP H . In addition, the absorption spectra of these intermediates were calculated based on the proposed reaction scheme. Together, these results indicate that the photocycle of PYP at room temperature has a branched pathway in the early stage and is essentially similar to that observed under low-temperature spectroscopy. Photoactive yellow protein (PYP) 1 is a bright-yellow protein found in the purple phototrophic bacterium Ec- tothiorhodospira halophila (1). It is proposed to be a photoreceptor protein for the negative phototaxis of the bacterium (2), and now similar proteins have been identified in various other organisms (3-6). PYP has a p-coumaric acid binding to the cysteine residue at position 69 by a thioester bond as its chromophore (7-9). The absorption maximum is located at 446 nm. The chromophore is isom- erized from the trans to the cis form on photon absorption (10-13), and PYP undergoes a photocycle (14-22) in which protein conformational change, proton transfer from Glu-46 to chromophore, and thermal re-isomerization of the chro- mophore take place. PYP is a small water-soluble protein of 14 kDa, and the tertiary structures of its intermediates (11-13) as well as the ground state have been analyzed (23). Due to these advantages, PYP is thought to be the most suitable target to understand the light-capturing mechanism of a photoreceptor protein. In particular, isomerization of the chromophore is the initial event upon photon absorption and is thought to be the key reaction to realize the highly effective utilization of photon energy for the photoreceptor proteins. To understand this primary process, the identifica- tion of the photocycle intermediates using ultrafast spec- troscopy is considered essential. The photochemistry of PYP has been extensively studied by flash photolysis at room temperature (14-19) and low-temperature spectroscopy (20, 21). As a result, several intermediates appearing in the photo- cycle have been identified thus far. The spectroscopic and kinetic properties of these intermediates are similar to those of the halobacterial retinal proteins, and the similarity in the mechanism of photoreactions between PYP and retinal pro- tein has been demonstrated. Ultrafast spectroscopy has pre- viously revealed that the stimulated emission of PYP on excitation with a laser pulse is composed of two components (τ ) 0.7, 3.6 ps) (17). Our femtosecond up-conversion ex- periment also revealed that the fluorescence decay curve is multiexponential and the decay time constant depends on the wavelength, suggesting the existence of multiple excited states (18). As a result of relaxation of the excited state, the first photoproduct called I0 (λ max ) 500 nm) is formed (19). I0 decays to I0q (λ max ) 500 nm), which is spectrally similar to but kinetically distinct from I0 (τ ) 220 ps). I0q decays to I1 (also called pR or PYP L )(λ max ) 460 nm, τ ) 3 ns), and subsequently decays to I2 (also called pB or PYP M )(λ max ) 350 nm, τ ) 500 μs). PYP M has the protonated chro- mophore unlike ground-state PYP, and the protein conforma- tion is largely altered. Finally, PYP is recovered from PYP M (τ ) 500 ms). † This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a SUNBOR grant, and by a grant from Kansai Research Foundation for Technology Promotion. * To whom correspondence should be addressed. Fax: 81 743 72 6109. E-mail: imamoto@ms.aist-nara.ac.jp. ‡ Nara Institute of Science and Technology. § Graduate School of Science, Osaka University. | Graduate School of Engineering, Osaka University. 1 Abbreviations: PYP, photoactive yellow protein; λmax, absorption maximum in the visible region; Tris, tris(hydroxymethyl)aminomethane; SVD, singular value decomposition; τ1/e, decay time constant. 6047 Biochemistry 2001, 40, 6047-6052 10.1021/bi002437p CCC: $20.00 © 2001 American Chemical Society Published on Web 04/26/2001