Photochemistry and Photobiology zyxwvutsrq Vol. 41, No. 6, pp. 681 zyxwvu - 688, 1985 Printed in Great Britain. zyxwvuts EDCBA All rights reserved 0031 zyx A -8655185 $03 zy .OO +O.OO Copyright zyx 0 1985 Pergamon Press Ltd THE KINETICS OF THE EARLY STAGES OF THE COMPARATIVE STUDY OF SMALL (60 kDalton) AND NATIVE (124 kDalton) PHYTOCHROMES FROM OAT PHYTOCHROME PHOTOTRANSFORMATION Pr -+ Pfr. A BELA P. RUZSICSKA, SILVIA E. BRASLAVSKY* and KURT SCHAFFNER Max-Planck-Institut fur Strahlenchemie, D-4330 Miilheim a. d. Ruhr, W. Germany (Received zyxwvu BA 14 November 1984; accepted 30 January 1985) Abstract-A comparative study of the decay kinetics of photogenerated transients from small (60 kDalton) and native (124 kDalton) oat phytochrome in the red-absorbing form (P,) in phosphate buffer containing 5 mM ethylenediamine tetraacetic acid, pH 7.8, (PB) and in PB containing 20% ethylene glycol, has been carried out in the temperature range 275-298 K. The analysis confirmed that at least two primary photoproducts, intermediates I& and Iym, are formed from P,. The kinetic parameters, as observed in PB at 695 nm and 275 K, are similar for the I;oo intermediates of both small and native phytochrome. Namely, the lifetimes are about 21 p,s (component percentages 38%) for the 1;""s and about 200 zyxwvutsrq ks (62%) for the I;bos. Arrhenius preexponential factors ( A ) of about loL6 and 1015 s-' and activation energies of about 61 and 56 kJ/mol were measured for the absorbance decays of the 1700s of small and native phytochrome, respectively. The kinetic data favour parallel paths for the formation of the I;,,,,s from P,, and the activation parameters indicate that the primary photoreactions of the transformation from P, to the far-red-absorbing form are restricted to the chromophore within the protein. Moreover, the relatively modest temperature dependence of the lifetimes of the I\,,,,s from small and native P, supports the working hypothesis that the ground state reactions to the ILL intermediates-although somewhat influenced by the polypeptide fragment that is removed upon degradation of native to small P,-are localized to the chromophore, as is most probably the case also for the primary photoreactions. The effect of the addition of 20% ethylene glycol on the pre-exponential factors of the time-dependent decay functions is discussed in similar terms of the early stages of the phototransformation. INTRODUCTION The spectral properties of native oat phytochrome (124 kDalton molecular weight; Quail el al., 1983; Litts et al., 1983), on the one hand, and of the degraded, but still photochromic, small (60 kDalton) and large (114 and 118 kDalton) oat phytochromes, on the other hand, have been reported to differ somewhat. The differences include a shift of the Pf,t absorption maximum from 730 nm for the native (this is identical with the zyxwvuts CBA in vivo Pf, maximum) to 722-725 nm for the smaller molecular sizes, and varying ratios A73dA070 for theabsorption spectrum at photo- equilibrium after saturating red-light irradiation (Furuya, 1983). Another important difference reported is the quantum yield for the P, + Pfr phototransformation, +,+f,, which is 1.5 times larger for native than for large P, (Vierstra and Quail, 1983a). These differences obviously originate somehow from the structural differences of the three *To whom correspondence should be addressed. TAbbreviutions: I,,,, intermediate absorbing at A,,, ca. 610 nm; I,,,,,, lumi-R, intermediate absorbing at A, , , ca. 700 nm; Ibl, meta-R, bleached intermediate; PB, 0.1 M phos- phate buffer + 5 rnM ethylenediamine tetraacetic acid solution, pH 7.8; P,, far-red-absorbing form of phy- tochrome; P,, red-absorbing form of phytochrome; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis. phytochromes. In other words, they reflect the influence of the proteinfragments, which are eliminatedin enzymatic degradation, on the chromophore properties and on the phototrans- formation kinetics. Our recent determination of the fluorescence yields and of the component-resolved picosecond fluorescence lifetimes of small and large (Wendler et al., 1984) and of native phytochromes (Holzwarth et al., 1984) has established that the main excited-state component in all three samples has the same lifetime, 48 k 3 ps. The fluorescence quantum yields (corrected for the percentage of the main lifetime component) are also very similar for all molecular sizes (1.5, 2.0 and 2.9 x for small, large, and native P,, respectively). This means that the sums of radiationless decays, which are directly related to the fluorescence lifetimes, must be equallysimilar. Moreover, their must be three orders of magnitude faster than the radiative decay. The difference in quantum yield for the P, + Pf, phototransformation of the different phytochromes may be due to different rate constants for the formation of the primary photoproduct (the first transient), which would be withinthe experimental errorof the measured lifetimes (Holzwarth et al., 1984). The phytochrome phototransformation, P, + Pf,, is discussed in terms of several intermediates. There 68 1