In Vitro Reconstitution of Light-harvesting POR-Protochlorophyllide Complex with Protochlorophyllides a and b

• Published in: The Journal of biological chemistry 2003-01, Vol.278 (2), p.807-815
• Main Authors: Reinbothe, Christiane, Buhr, Frank, Pollmann, Stephan, Reinbothe, Steffen
• Format: Article
• Language: English
• Subjects: Blotting, Western; Chromatography, High Pressure Liquid; Hordeum - metabolism; Light; Oxidoreductases - metabolism; Oxidoreductases Acting on CH-CH Group Donors; Plant Proteins - metabolism; Protochlorophyllide - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M209738200

protochlorophyllide oxidoreductase (POR; EC1.1.33.1) is a key enzyme for the light-induced greening of angiosperms. In barley, two POR proteins exist, termed PORA and PORB. These have previously been proposed to form higher molecular weight light-harvesting complexes in the prolamellar body of etioplasts (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999)Nature 397, 80–84). Here we report the in vitro reconstitution of such complexes from chemically synthesized protochlorophyllides (Pchlides) a andb and galacto- and sulfolipids. Low temperature (77 K) fluorescence measurements revealed that the reconstituted, lipid-containing complex displayed the same characteristics of photoactive Pchlide 650/657 as the presumed native complex in the prolamellar body. Moreover, Pchlide F650/657 was converted to chlorophyllide (Chlide) 684/690 upon illumination of the reconstituted complex with a 1-ms flash of white light. Identification and quantification of acetone-extractable pigments revealed that only the PORB-bound Pchlide a had been photoactive and was converted to Chlide a, whereas Pchlide b bound to the PORA remained photoinactive. Nondenaturing PAGE of the reconstituted Pchlide a/b-containing complex further demonstrated a size similar to that of the presumed native complexin vivo, suggesting that both complexes may be identical.