PHOTOREACTION CYCLE OF PHOTOACTIVE YELLOW PROTEIN FROM ECTOTHIORHODOSPIRA-HALOPHILA STUDIED BY LOW-TEMPERATURE SPECTROSCOPY

The photocycle of photoactive yellow protein (PYP) from Ectothiorhodos pira halophila was studied by low-temperature spectroscopy. Irradiatio n of PYP at -190 degrees C produced a photo-steady-state mixture compo sed of bathochromic and hypsochromic photoproducts (PYPB and PYPH). Up on warming, PYPH was thermally converted to a slightly blue-shifted in termediate (PYPHL) above -150 degrees C and then to a red-shifted one (PYPL) above -80 degrees C. PYPB was thermally converted to the blue-s hifted intermediate (PYPBL) above -180 degrees C and then to PYPL abov e -90 degrees C. PYPL thermally reverted to PYP above -50 degrees C, c ompleting the photocycle. The spectral properties of PYPL formed at lo w temperature suggest that it corresponds to the red-shifted photoprod uct detected in the nano- to microsecond time scale at room temperatur e (A(465)) The absolute absorption spectra of PYPH, PYPB, and PYPL wer e 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. A lthough a near-UV intermediate (A(355)) is observed in the recovery pr ocess of PYP from A(465) at room temperature, it was not detected at l ow temperatures, probably due to the effects of temperature and the pr esence of glycerol. A scheme of the photocycle of PYP is presented.