The photocycle of photoactive yellow protein (PYP) from Ectothiorhodospira halophila was studied by low-temperature spectroscopy. Irradiation of PYP at -190 degrees C produced a photo-steady-state mixture composed of bathochromic and hypsochromic photoproducts (PYPB and PYPH). Upon warming, PYPH was thermally converted to a slightly blue-shifted intermediate (PYPHL) above -150 degrees C and then to a red-shifted one (PYPL) above -80 degrees C. PYPB was thermally converted to the blue-shifted intermediate (PYPBL) above -180 degrees C and then to PYPL above -90 degrees C. PYPL thermally reverted to PYP above -50 degrees C, completing the photocycle. The spectral properties of PYPL formed at low temperature suggest that it corresponds to the red-shifted photoproduct detected in the nano- to microsecond time scale at room temperature (A465). The absolute absorption spectra of PYPH, PYPB, and PYPL were estimated, and their absorption maxima were determined to be 442 and 489 nm at -190 degrees C and 456 nm at -80 degrees C, respectively. Although a near-UV intermediate (A355) is observed in the recovery process of PYP from A465 at room temperature, it was not detected at low temperatures, probably due to the effects of temperature and the presence of glycerol. A scheme of the photocycle of PYP is presented.

The gene encoding photoactive yellow protein (PYP) was isolated from Ectothiorhodospira halophila, and a high-level expression system for PYP was constructed in Escherichia coli. The molecular weight and the absorption spectrum of PYP expressed in E. coli were identical with those of the native PYP isolated from E. halophila. The amino acid residues which might interact with the chromophore (Tyr42, Glu46, Thr50, Arg52, and Cys69) were mutated by site-directed mutagenesis and the absorption spectra of these mutants were examined to study the chromophore/protein interaction in PYP. The former three substitutions (Y42F, E46Q, and T50V) brought about red-shifts of the absorption spectra, but the substitution of Arg52 (R52Q) brought about no change and that of Cys69 (C69S) led to no formation of pigments. These results suggest that Tyr42, Glu46, and Thr50 strongly interact with the chromophore, while Arg52 does not contribute the color tuning of PYP.

The photocycle intermediates of photoactive yellow protein (PYP) were characterized by low-temperature Fourier transform infrared spectroscopy. The difference FTIR spectra of PYP(B), PYP(H), PYP(L), and PYP(M) minus PYP were measured under the irradiation condition determined by UV-visible spectroscopy. Although the chromophore bands of PYP(B) were weak, intense sharp bands complementary to the 1163-cm(-1) band of PYP, which show the chromophore is deprotonated, were observed at 1168-1169 cm(-1) for PYP(H) and PYP(L), indicating that the proton at Glu46 is not transferred before formation of PYP(M). Free trans-p-coumaric acid had a 1294-cm(-1) band, which was shifted to 1288 cm(-1) in the cis form. All the difference FTIR spectra obtained had the pair of bands corresponding to them, indicating that all the intermediates have the chromophore in the cis configuration. The characteristic vibrational modes at 1020-960 cm(-1) distinguished the intermediates. Because these modes were shifted by deuterium-labeling at the ethylene bond of the chromophore while labeling at the phenol part had no effect, they were attributed to the ethylene bond region. Hence, structural differences among the intermediates are present in this region. Bands at about 1730 cm(-1), which show that Glu46 is protonated, were observed for all intermediates except for PYP(M). Because the frequency of this mode was constant in PYP(B), PYP(H), and PYP(L), the environment of Glu46 is conserved in these intermediates. The photocycle of PYP would therefore proceed by changing the structure of the twisted ethylene bond of the chromophore.