Biosynthesis of a chlorophyllide b-like pigment in phenanthroline-treated Chlamydomonas reinhardtii y-1

Incubation of degreened Chlamydomonas reinhardtii y-1 cells in the dark with m-phenanthroline induced de novo synthesis of a chlorophyllide b-like pigment. The rate of synthesis of this pigment in the dark was greater than that of total chlorophyll in illuminated cells. Most of the newly synthesized pigment was excreted into the culture medium. The product was extracted from the medium as the metal-free pheophorbide, which had a fluorescence excitation maximum at 428 +/- 1 nm and an emission maximum at 657 +/- 1 nm (E428F657) in ethyl acetate (E427F657 in diethyl ether). Three pheophorbide species were extracted from the medium of green cells treated in the dark, a minor component with a spectrum (E410F670) identical to demetallated chlorophyll a, and two major species with spectral values of E428F657 and E433F657. The latter, predominant form had a spectrum identical to demetallated chlorophyll b, which was purified from the algal cells. E428F657 and E433F657 reacted with hydroxylamine and Girard's T-reagent, which caused a shift in the fluorescence emission maximum to 668 nm. Pheophytin b, which contains an aldehyde group, exhibited an identical spectral shift when treated in the same way, but pheophytin a or porphyrin biosynthetic intermediates did not. Proton NMR analysis of the E428F657 chlorin produced by yellow cells treated with m-phenanthroline confirmed the presence of an aldehydic proton. Chelating and nonchelating phenanthroline analogs equally stimulated synthesis of this product.