Time-Resolved Absorption Studies of Native Etiolated Oat Phytochrome

The phototransformation kinetics of native oat phytochrome from etiolated seedling shoots of Avena sativa was investigated by laser photolysis experiments at 10 °C. The phototransformation from the red-absorbing form (Pr) to the far-red-absorbing form (Pfr) was initiated by 7-ns (fwhm) laser pulses at 638 nm. The transient absorption difference spectra in the UV and visible regions were recorded from 100 ns to 800 ms after photolysis by a gated optical multichannel analyzer system. A global fitting procedure employing a singular value decomposition method was used to simultaneously analyze the difference spectra at various times after photolysis. The global analysis of the laser photolysis data shows that the phototransformation from Pr to Pfr involves five kinetic intermediates. At 10 °C the apparent lifetimes associated with these intermediates are 7.4 μs, 89.5 μs, 7.6 ms, 42.4 ms, and at least 266 ms. It is possible to fit the transient absorption data with kinetic models involving two parallel pathways or sequential pathways with equilibria at certain stages of the phototransformation. The formation of Pfr involves an unbranched reaction. Discussions of the possible kinetic mechanisms of the phototransformation and the absorption spectra of the intermediates derived from these models are presented.