Photochemistry and Photobiology, 1998, 68(5): 754-761 zyxwvut The Complexity of the P, to P,, Phototransformation Kinetics Is an Intrinsic Property of Native Phytochrome* Peter Schmidt, Thomas Gensch, Anja Remberg, Wolfgang Gartner, Silvia E. Braslavskyt and Kurt Schaffner Max-Planck-lnstitut fur Strahlenchemie, Mulheim an der Ruhr, Germany Received 17 February 1998; accepted 21 August 1998 ABSTRACT zyxwvutsrq Several possible origins of the complex phytochrome red to far-red light-absorbing phytochrome (P, -+ P,) pho- totransformation kinetics in the nanosecond-to-second time range have been examined. Heterogeneity based on protein sequence is ruled out as an origin of the multi- component kinetics because recombinant 124 kDa oat phytochrome A apoprotein reconstituted with phytochro- mobilin and the native protein are very similar in this regard throughout this time range. The P, forms of na- tive 124 kDa oat phytochrome A and of a homogeneous recombinant 65 kDa chromoprotein fragment exhibit thermochromic properties interpreted as arising in each case from the presence of two P, species in thermal equi- librium. They exhibit identical photochemical properties. The complex kinetics therefore cannot result from P, het- erogeneity either. Thus, the presence of two P, forms in equilibrium zyxwvutsr (Pr,675 and P,,& and the complex multiex- ponential P, -+ Pf, phototransformation kinetics observed in all time ranges are intrinsic properties of the homo- geneous holoprotein of oat phytochrome A. INTRODUCTION zyxwvutsrq Phytochromes constitute a family of chromoproteins ( 1-3) that consist of a protein moiety of -124 kDa (about 1100 amino acid residues), to which the bilatriene phytochromo- hilin (PQB) is covalently bound through a thioether bond. Phytochrome A (phyA), the phytochrome variety mainly present in dark-grown plants zyxwvutsrqp (vide zyxwvutsrqp infru), exists in solution as a dimer (4-6). Phytochromes assume two stable and spectrally distinct “This paper is dedicated to the memory of Juanjo Cosa, deceased on 28 September 1997. ?To whom correspondence should be addressed at: Max-Planck- Institut fur Strahlenchemie, Postfach 10 13 65, D-45413 Mulheim an der Ruhr, Germany. Fax: xx49-208-306-395 1 ; e-mail: braslavskys@mpi-muelheim.mpg.de $Abbreviations: CW, continuous-wave; dc, direct current; LADS, lifetime-associated difference spectrum: PCB, phycocyanobilin; phyA, phytochrome A (predominant form in dark-grown plants); P,, P,,, red and far-red light-absorbing, thermally stable forms of phytochrome, respectively; PQB, phytochomobilin; SDS, sodium dodecyl sulfate. tl 1998 American Society for Photobiology 003 1-8655/98 zyxwvutsrqpo $S.00+0.00 forms: red (P,) and far-red light-absorbing phytochrome (P,) with A , around 667 and -730 nm, respectively, the precise position depending on the phytochrome type. Both states are interconverted by irradiation with light of the appropriate wavelength. The photochromic phytochrome system, P, S P,,, thus functions as a light-driven biological switch, adopt- ing a photoequilibrium position determined by the environ- mental light conditions. The P, 4 Pf, phototransformation kinetics in the micro- second-to-second time window has been studied extensively for phyA from oat (Avena zyxw sutivu L.) and from other plants (for a review see Sineshchekov (7)). Parallel and sequential models and schemes including equilibration of intermediates have been proposed to rationalize the complex kinetics ob- served (8-1 1). This paper investigates two possible kinds of heteroge- neity in phyA and the influence on the photoinduced reac- tions: (1) amino acid sequence heterogeneity and (2) differ- ent conformations of P, in thermal equilibrium. In higher plants there exist several types of phytochromes (phyA, phyB, phyC, etc.) with different functions. Their amino acid sequences vary by up to 50% (12). Even the same type, e.g. oat phyA, is expressed in plants with at least three different sequences (AP 3 , 4 and 5) that possess a 98% homology (13,14). The relative contributions of the three proteins in oat are -0.6:1:1. It has been speculated that the variation of the amino acid sequences of AP 3, 4 and 5 be responsible for multiexponential thermal reversions from Pf, to P, and for the complexity of the P, -+ P,, transformation kinetics (13). Phytochrome heterogeneity may also be indicated by a temperature dependence of the P, absorption spectrum. Changes of the spectra of P, and Pf, and of the phototrans- formation intermediates with temperature have already been observed by Burke et ul. (15) and by Rudiger and Thummler (16) in low-temperature studies. Furthermore, Sasaki et al. (17) found a general increase in absorbance with decreasing temperature concomitant with a red shift of the P, and Pf, maxima in partially degraded (1 14 kDa) pea phyA between -110 and 30°C. We shall address similar changes in full- length oat phyA. Another possible heterogeneity in phytochrome is unrav- elled by analyzing the thermal Pf, -+ P, reversion. This re- action takes place in vitro in time ranges from minutes to days, depending on phytochrome type, origin and size. Vier- 754