In vitro reconstitution of light-harvesting POR-protochlorophyllide complex with protochlorophyllides a and b.
NADPH:protochlorophyllide oxidoreductase (POR; EC ) 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 and b 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 complex in vivo, suggesting that both complexes may be identical.
Reinbothe
Christiane
C
Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, Universitätsstrasse 30, Germany. christiane.reinbothe@uni-bayreuth.de
Buhr
Frank
F
Pollmann
Stephan
S
Reinbothe
Steffen
S
D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D001467:Hordeum / Q000378:metabolism*; D008027:Light; D010088:Oxidoreductases / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010940:Plant Proteins / Q000378:metabolism*; D011521:Protochlorophyllide / Q000378:metabolism*
D010940:Plant Proteins; D011521:Protochlorophyllide; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase