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J. Biol. Chem., Vol. 279, Issue 43, 44400-44406, October 22, 2004

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Early Steps in the Assembly of Light-harvesting Chlorophyll a/b Complex

TIME-RESOLVED FLUORESCENCE MEASUREMENTS^*

Ruth Horn and Harald Paulsen

From the Institut für Allgemeine Botanik der Johannes-Gutenberg-Universität, Müllerweg 6, D-55099 Mainz, Germany

The light-harvesting chlorophyll a/b complex (LHCIIb) spontaneously assembles from its pigment and protein components in detergent solution. The formation of functional LHCIIb can be detected in time-resolved experiments by monitoring the establishment of excitation energy transfer from protein-bound chlorophyll b to chlorophyll a. To detect the possible initial steps of chlorophyll binding that may not yet give rise to chlorophyll b-to-a energy transfer, we have monitored LHCIIb assembly by measuring excitation energy transfer from a fluorescent dye, covalently bound to the protein, to the chlorophylls. In order to exclude interference of the dye with protein folding or pigment binding, the experiments were repeated with the dye bound to four different positions in the protein. Initial chlorophyll binding occurs at roughly the same rate as the establishment of chlorophyll b-to-a energy transfer, in the range of 10 s. However, under limiting chlorophyll concentrations, the binding of chlorophyll a clearly precedes that of chlorophyll b. The complex containing the apoprotein, carotenoids, and chlorophyll a but no chlorophyll b is biochemically unstable and therefore cannot be isolated. However, chlorophyll a binding into this weak complex is specific, as it does not occur with a C-terminal deletion mutant of Lhcb1 which still contains most chlorophyll-ligating amino acids but is unable to fold and assemble into functional LHCIIb. As a scenario for LHCIIb assembly in the thylakoid, we propose the initial formation of a labile Lhcb1-chlorophyll a-carotenoid complex that then becomes stabilized by the binding (or formation in situ) of chlorophyll b.

Received for publication, June 28, 2004 , and in revised form, August 5, 2004.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant Pa 324/3-3,4. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-6131-3924633; Fax: 49-6131-3923787; E-mail: paulsen{at}mail.uni-mainz.de, rhorn{at}mail.uni-mainz.de.

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