THE KINETICS OF THE EARLY STAGES OF THE PHYTOCHROME PHOTOTRANSFORMATION P_r→ P_fr. A COMPARATIVE STUDY OF SMALL (60 kDalton) and NATIVE (124 kDalton) PHYTOCHROMES FROM OAT

Abstract

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 (Pr) 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 Ii700s and Ii7oo are formed from Pr. The kinetic parameters, as observed in PB at 695 nm and 275 K, are similar for the I700 intermediates of both small and native phytochrome. Namely, the lifetimes are about 21 μs (component percentages 38%) for the I Ii700s and about 200 μ.s (62%) for the Ii700S‐ Arrhenius preexponential factors (A) of about 1016 and 1015 s‐1and activation energies of about 61 and 56 kJ/mol were measured for the absorbance decays of the I700S of small and native phytochrome, respectively. The kinetic data favour parallel paths for the formation of the Ii700s from Pr, and the activation parameters indicate that the primary photoreactions of the transformation from Pr 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 Ii700S from small and native Pr supports the working hypothesis that the ground state reactions to the Iibl, intermediates–although somewhat influenced by the polypeptide fragment that is removed upon degradation of native to small Pr–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.

Resonance Raman spectroscopic study of the tryptic 39‐kDa fragment of phytochrome

“…Still, recombinant and tryptic phytochrome fragments, that range either from amino acid residues 1 to 595 (65 kDa) or 65 to 595 (59 kDa) only, i.e. lack the C‐terminal part, display full photoreversibility and kinetics of the P r /P fr photointerconversion similar to the wild‐type photoreceptor [4–6], in accord with the observation that the chromophore structure and the photoinduced structural changes of the recombinant 65‐kDa phytochrome are essentially the same as well [7,8]. However, truncation at position 425 by tryptic digestion substantially affects the formation of P fr , as indicated by the intensity reduction of the long‐wavelength absorption band.…”

Intermediates of reversible photochemistry of phycoerythrocyanin α from Mastigocladuslaminosus probed by low temperature absorptionand circular dichroism spectroscopy

“…The chromophore-pocket is expected to be more open in α-PEC, which may add to the strong influence of the solvent. By contrast, photochemistry of phytochrome hardly depends on solvent viscosity (Ruzsicska et al, 1985), but rather appears to depend more on internal modes of the protein. It already shows changes at 205 K (Kendrick and Spruit, 1977), but even below 175 K, phytochrome can be photo-driven effectively (> 50%) to the first intermediate (Eilfeld and Rüdiger, 1985), which has probably already undergone Z/E isomerization (Eilfeld and Eilfeld, 1988).…”

Phototransformation of oat phytochrome with millisecond and submillisecond flashes: The level of the photostationary Pfr/Ptot ratio is modified by photoreversible intermediates

“…However, the late intermediate, I|,|, which according to recent results is also photoreversible (Pratt et al 1984) cannot yet be excluded definitely. On the other hand, it seems unlikely that the very early intermediate I^ (lumi-R), which was recently postulated to be photoreversible within nanosecond flashes in vitro (Jabben et al 1984) as in vivo (Scheuerlein and Braslavsky 1985), affects the photostationary equilibrium 4> for repetitive ms-flashes substantially, because its lifetime is in the early microsecond range (Ruzsicska et al 1985).…”