Analysis of Phototransformation Intermediates in the Pathway From the Red‐absorbing to the Far‐red‐absorbing Form of Avena Phytochrome by a Multichannel Transient Spectrum Analyzer

Phototransformation of the red‐absorbing form of phytochrome (Pr) to the far‐red‐absorbing form (Pfr) was followed with a custom‐built transient spectrum analyzer. Large phytochrome, which consisted of approximately 120000‐dalton monomers, was immunopurified or conventionally purified from etiolated oat (Avena sativa L., cv. Garry) shoots. Phototransformation was initiated by exciting Pr with a 115‐mJ, 600‐ns half‐width, 655‐nm laser pulse. Absorption spectra were recorded on a microsecond time scale at predetermined times after the flash. It has been reported earlier that flash excitation of large oat Pr produces a transformation intermediate with maximum absorbance near 700 nm in a difference spectrum and that this intermediate decays by two kinetically distinct reactions. Difference spectra for these two reactions are indistinguishable. Both show bleaching centered at 690 nm with no detectable associated absorbance increase between 570 and 830 nm. Subsequent appearance of absorbance at 724 nm, which presumably but not necessarily represents the appearance of Pfr, had earlier been shown to occur by two kinetically distinct reactions for large oat phytochrome. Data presented here indicate in addition the occurrence of a third, slower reaction. Difference spectra for the two faster reactions are indistinguishable, both with maxima near 728 nm and minima near 650 nm. The difference spectrum for the slowest component, however, was qualitatively different exhibiting a maximum near 722 nm with no corresponding minimum. About 15‐20% of the absorbance increase at 724 nm occurred by this slowest reaction, which exhibited a half‐life of 3 s at 25°C and a Q10 of 1.2 for immunopurified and 1.5 for conventionally purified phytochrome. The percentage occurring by this reaction was independent of temperature over the range studied (1‐25dEC). For immunopurified phytochrome the enthalpy of activation, Gibbs free energy of activation, and entropy of activation of this slowest reaction were found to be about lOkJ‐mol‐1, 75kJ.mol‐1, and ‐220 J.mol‐1 K‐1, respectively, and for conventionally purified phytochrome 25kJ.mol‐1, 75kJ.mol‐1and —170 J.mol‐1 K‐1, respectively. The thermodynamic characteristics of this reaction indicate that it may involve a significant ordering of the protein moiety as it transforms to Pfr.