Synthesis of chlorophyll b: localization of chlorophyllide a oxygenase and discovery of a stable radical in the catalytic subunit.
BACKGROUND
Assembly of stable light-harvesting complexes (LHCs) in the chloroplast of green algae and plants requires synthesis of chlorophyll (Chl) b, a reaction that involves oxygenation of the 7-methyl group of Chl a to a formyl group. This reaction uses molecular oxygen and is catalyzed by chlorophyllide a oxygenase (CAO). The amino acid sequence of CAO predicts mononuclear iron and Rieske iron-sulfur centers in the protein. The mechanism of synthesis of Chl b and localization of this reaction in the chloroplast are essential steps toward understanding LHC assembly.


RESULTS
Fluorescence of a CAO-GFP fusion protein, transiently expressed in young pea leaves, was found at the periphery of mature chloroplasts and on thylakoid membranes by confocal fluorescence microscopy. However, when membranes from partially degreened cells of Chlamydomonas reinhardtii cw15 were resolved on sucrose gradients, full-length CAO was detected by immunoblot analysis only on the chloroplast envelope inner membrane. The electron paramagnetic resonance spectrum of CAO included a resonance at g = 4.3, assigned to the predicted mononuclear iron center. Instead of a spectrum of the predicted Rieske iron-sulfur center, a nearly symmetrical, approximately 100 Gauss peak-to-trough signal was observed at g = 2.057, with a sensitivity to temperature characteristic of an iron-sulfur center. A remarkably stable radical in the protein was revealed by an isotropic, 9 Gauss peak-to-trough signal at g = 2.0042. Fragmentation of the protein after incorporation of 125I- identified a conserved tyrosine residue (Tyr-422 in Chlamydomonas and Tyr-518 in Arabidopsis) as the radical species. The radical was quenched by chlorophyll a, an indication that it may be involved in the enzymatic reaction.


CONCLUSIONS
CAO was found on the chloroplast envelope and thylakoid membranes in mature chloroplasts but only on the envelope inner membrane in dark-grown C. reinhardtii cells. Such localization provides further support for the envelope membranes as the initial site of Chl b synthesis and assembly of LHCs during chloroplast development. Identification of a tyrosine radical in the protein provides insight into the mechanism of Chl b synthesis.
Eggink
Laura L
LL
School of Life Sciences, Arizona State University, Tempe, Arizona 85287-4501, USA. leggink@asu.edu
LoBrutto
Russell
R
Brune
Daniel C
DC
Brusslan
Judy
J
Yamasato
Akihiro
A
Tanaka
Ayumi
A
Hoober
J Kenneth
JK
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism; D002734:Chlorophyll / Q000096:biosynthesis*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005609:Free Radicals / Q000737:chemistry; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D007454:Iodides / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018532:Pisum sativum / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020524:Thylakoids / Q000378:metabolism; D014162:Transfection
D005609:Free Radicals; D007454:Iodides; D007506:Iron-Sulfur Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D002734:Chlorophyll; D049452:Green Fluorescent Proteins; C037184:chlorophyll b; D010105:Oxygenases; C115272:chlorophyll a oxygenase