PHOTOINDUCED VOLUME CHANGE AND ENERGY-STORAGE ASSOCIATED WITH THE EARLY TRANSFORMATIONS OF THE PHOTOACTIVE YELLOW PROTEIN FROM ECTOTHIORHODOSPIRA-HALOPHILA

The photocycle of the photoactive yellow protein (PYP) isolated from E ctothiorhodospira halophila was analyzed by flash photolysis with abso rption detection at low excitation photon densities and by temperature -dependent laser-induced optoacoustic spectroscopy (LIOAS). The quantu m yield for the bleaching recovery of PYP, assumed to be identical to that for the phototransformation of PYP (pG), to the red-shifted inter mediate, pR, was phi(R) = 0.35 +/- 0.05, much lower than the value of 0.64 reported in the literature. With this value and the LIOAS data, a n energy content for pR of 120 kJ/mol was obtained, similar to 50% low er than for excited pG. Concomitant with the photochemical process, a volume contraction of 14 ml/photoconverted mol was observed, comparabl e with the contraction (11 ml/mol) determined for the bacteriorhodopsi n monomer. The contraction in both cases is interpreted to arise from a protein reorganization around a phototransformed chromophore with a dipole moment different from that of the initial state. The deviations from linearity of the LIOAS data at photon densities >0.3 photons per molecule are explained by absorption by pG and pR during the laser pu lse duration (i.e., a four-level system, pG, pR, and their respective excited states). The data can be fitted either by a simple saturation process or by a photochromic equilibrium between pG and pR, similar to that established between the parent chromoprotein and the first inter mediate(s) in other biological photoreceptors. This nonlinearity has i mportant consequences for the interpretation of the data obtained from in vitro studies with powerful lasers.