J. Photochem. Photobiol. B: Biol., 18 (1993) 17-25 17 Picosecond events in the phototransformation of phytochrome - a time-resolved absorption study? Max E. Lippitsch”,++, Gudrun Hermannb, Harald Brunnef, Eberhard Miillerb and Franz R. Aussenegg” “Institut ji’ir Experimentalphysik, Karl-Franzens-Universitiit Graz, Universitiitsplatz 5, A-8010 Graz (Austria) bBiologische Fakutiit, Friedrich-Schiller-Universitiit Jena, Philosophenweg 12, O-6900 Jena (Germany) zyxwvutsrqponmlkjihgfedcbaZYX (Received July 31, 1992; accepted September 30, 1992) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Abstract Picosecond time-resolved transient absorption studies with phytochrome in its red light absorbing form (Pr) isolated from rye (Se&e cereale cv. Danae) are reported. After excitation of P, with 6 ps pulses at 621 nm the transient absorption spectra were measured at various delay times from 5 ps up to 1 ns. Upon excitation an increase in the transient absorption around 400 nm immediately appeared, which is ascribed to the first excited state absorption of P,. With a delay of about 15 ps a strong bleaching of the P, ground state absorption around 665 nm emerged. After a series of further absorption changes, an increase in absorbance occurred above 685 nm. Since this transient absorption persisted over the 1 ns time-window measured, it is interpreted as being due to the formation of lumi-R. Based on the time evolution of the observed absorption changes it seems likely that two pools of lumi-R were formed. In addition, it was found from the kinetics of the picosecond absorption changes that the relaxation of excited P, obviously involves processes which follow a non-exponential rate law. From a comparison with picosecond adsorption spectroscopy on deuterated P, and a 39 kDa chromopeptide, conclusions about the first relaxation steps in the phototransformation of P, are drawn. It is suggested that they involve rotations at the single bonds in the chromophoric methine bridges. Keywords: Phytochrome, picosecond excitation, lumi-R, phototransformation 1. Introduction Phytochrome is known to regulate a great di- versity of photomorphogenic responses in higher plants. Photomorphogenic control occurs via the photoreversible transformation between the phys- iologically inactive red light absorbing form of phytochrome (P,) and the physiologically active far-red light absorbing form (P,J. The molecular mechanism involved in this phototransformation is the subject of intense investigations since the discovery of phytochrome in 1959 [l]. Proton trans- location, Z, E isomerization and conformational changes within the chromophore or a combination of these processes have been discussed as adequate models for the primary reaction following im- mediately the the absorption of light. +Dedicated to Professor Dr K. Schaffner on the occasion of his 60th birthday. ++Authorto whom correspondence should be addressed. Results recently obtained from comparative spectroscopic studies with chromopeptides of phy- tochrome and phycocyanin provide strong evidence that the primary photoreaction is a Z, E isomer- ization around the Cl5 methine bridge of the tetrapyrrolic chromophore [2, 31. Although this model has been widely accepted, there remain unresolved questions concerning the detailed in- sight into the underlying mechanism. In order to address these questions time-resolved spectro- scopic techniques are a valuable tool. In earlier studies, flash photolysis techniques with micro- and nanosecond time resolution were employed [4-151. They provided important knowl- edge about the sequence of events leading from P, to Pr, and vice versa, but the primary events initiated by the absorption of light still remained unclear. Since these events are expected to proceed within picoseconds, they require ultrafast spec- troscopic techniques for their analysis. This fact has stimulated a number of ultrafast time-resolved studies (for a review, see ref. 16) loll-1344/93/$6.00 0 1993 - Elsevier Sequoia. All rights reserved