Recombinant phytochrome of the moss Ceratodan purpureus: Heterologous expression and kinetic analysis of P-r -> P-fr conversion

The phytochrome-encoding gene Cerpu;PHY;2 (CP2) of the moss Ceratodon purpu reus was heterologously expressed in Saccharomyces cerevisiae as a polyhist idine-tagged apoprotein and assembled with phytochromobilin (P Phi B) and p hycocyanobilin (PCB), Nickel-affinity chromatography yielded a protein frac tion containing approximately 80% phytochrome, The holoproteins showed phot oreversibility with both chromophores, Difference spectra gave maxima at 64 4/716 nm (red-absorbing phytochrome [P-r]/far-red-absorbing phytochrome [P- fr]) for the PCB adduct, and 659/724 nm for the P Phi B-adduct, the latter in close agreement with values for phytochrome extracted from Ceratodon its elf, implying that P Phi B is the native chromophore in this moss species, Immunoblots stained with the antiphytochrome antibody APC1 showed that the recombinant phytochrome had the same molecular size as phytochrome from Cer atodon extracts. Further, the mobility of recombinant CP2 holophytochrome o n native size-exclusion chromatography was similar to that of native oat ph ytochrome, implying that CP2 forms a dimer, Kinetics of absorbance changes during the P-r --> P-fr photoconversion of the PCB adduct, monitored betwee n 620 and 740 nm in the microsecond range, revealed the rapid formation of a red-shifted intermediate (I-700), decaying with a time constant of simila r to 110 mu s. This is similar to the behavior of phytochromes from higher plants when assembled with the same chromophore, When following the formati on of the P-fr state, two major processes were identified (with time consta nts of 3 and 18 ms) that are followed by slow reactions in the range of 166 ms and 8 s, respectively, albeit with very small amplitudes.