Synthesis of chlorophyll b: Localization of chlorophyllide a oxygenase and discovery of a stable radical in the catalytic subunit

被引：52

作者：

Eggink L.L. ^{[1
,3
]}

LoBrutto R. ^{[1
,3
]}

Brune D.C. ^{[2
,3
]}

Brusslan J. ^{[4
]}

Yamasato A. ^{[5
]}

Tanaka A. ^{[5
]}

Hoober J.K. ^{[1
,3
]}

机构：

[1] School of Life Sciences, Arizona State University, Tempe

[2] Dept. of Chemistry and Biochemistry, Arizona State University, Tempe

[3] Ctr. Stud. Early Events P., Arizona State University, Tempe

[4] Department of Biological Science, California State University, Long Beach

[5] Institute of Low Temperature Science, Hokkaido University

来源：

BMC Plant Biology | / 4卷 / 1期

基金：

美国国家科学基金会;

关键词：

Thylakoid Membrane; Chloroplast Development; Envelope Membrane; Chloroplast Envelope; Chlamydomonas Cell;

D O I：

10.1186/1471-2229-4-5

中图分类号：

学科分类号：

摘要：

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. Conclusion: 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. © 2004 Eggink et al; licensee BioMed Central Ltd.

引用

页数：16

共 64 条

[21]

White R.A., Wolfe G.R., Komine Y., Hoober J.K., Localization of light-harvesting complex apoproteins in the chloroplast and cytoplasm during greening of Chlamydomonas reinhardtii at 38°C, Photosynth Res, 47, pp. 267-280, (1996)

[22]

Bovet L., Muller M.O., Siegenthaler P.A., The 26- and 14-kDa phosphoproteins associated with spinach chloroplast envelope membranes are distinct membrane-bound poosl of the light-harvesting complex of photosystem II and of the small subunit of the ribulose-1,5- bisphosphate carboxylase-oxygenase, Planta, 195, pp. 563-569, (1995)

[23]

Bovet L., L'Eplattenier B., Siegenthaler P.A., In vitro and in organello phosphorylation of envelope proteins and phosphoglucomutase in spinach chloroplasts, Plant Sci, 128, pp. 169-180, (1997)

[24]

Wingfield P.T., Production of recombinant proteins, Current Protocols in Protein Science Volume 1, 1, pp. 501-5318, (2000)

[25]

Schmidt C.L., Hatzfeld O.M., Petersen A., Link T.A., Schafer G., Expression of the Solfolobus acidocaldarius Rieske iron sulfur protein II (SOXF) with the correctly inserted [2Fe-2S] cluster in Escherichia coli, Biochem Biophys Res Commun, 234, pp. 283-287, (1997)

[26]

Hagerhall C., Magnitsky S., Sled V.D., Schroder I., Gunsalus R.P., Cecchini G., Ohnishi T., An Escherichia coli mutant quinol:fumarate reductase contains an EPR-detectable semiquinone stabilized at the proximal quinone-binding site, J Biol Chem, 274, pp. 26157-26164, (1999)

[27]

Basu P., Katterle B., Andersson K.K., Dalton H., The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein, Biochem J, 369, pp. 417-427, (2003)

[28]

Knowles P.F., Brown III R.D., Koenig S.H., Wang S., Scott R.A., McGuirl M.A., Brown D.E., Dooley D.M., Spectroscopic studies of the active site of galactose oxidase, Inorg Chem, 34, pp. 3895-3902, (1995)

[29]

Klinman J.P., Mechanisms whereby mononuclear copper proteins functionalize organic substrates, Chem Rev, 96, pp. 2541-2561, (1996)

[30]

Roberts A.G., Bowman M.K., Kramer D.M., Certain metals are inhibitors of cytochrome b<sub>6</sub>f complex 'Rieske'iron-sulfur protein domain movements, Biochemistry, 41, pp. 4070-4079, (2002)

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