Primary photoreaction of photoactive yellow protein studied by subpicosecond-nanosecond spectroscopy.

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 degrees 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.