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

The primary photochemical event of photoactive yellow protein (PYP) was stu died by laser flash photolysis experiments on a subpicosecond-nanosecond ti me scale. PYP was excited by a 390-nm pulse, and the transient difference a bsorption spectra were recorded by a multichannel spectrometer for a more r eliable spectral analysis than previously possible. Just after excitation, an absorbance decrease due to the stimulated emission at 500 nm and photoco nversion 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 spec trum was formed after 2 ns and was stable for at least 5 ns. The latter is therefore believed to correspond to I1 (PYPL), which has been detected on a nanosecond time scale or trapped at -80 degreesC. Singular value decomposi tion 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 con version from PYPB to PYPL. The amount of PYPL at 5 ns was less than that of photoconverted PYP, suggesting the formation of another intermediate, PYPH . In addition, the absorption spectra of these intermediates were calculate d based on the proposed reaction scheme. Together, these results indicate t hat the photocycle of PYP at room temperature has a branched pathway in the early stage and is essentially similar to that observed under low-temperat ure spectroscopy.