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Interaction of bacteriochlorophyll with the LH1 and PufX polypeptides of photosynthetic bacteria: use of chemically synthesized analogs and covalently attached fluorescent probes

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Abstract

The protein components of the reaction center (RC) and core light-harvesting (LH 1) complexes of photosynthetic bacteria have evolved to specifically, but non-covalently, bind bacteriochlorophyll (Bchl). The contribution to binding of specific structural elements in the protein and Bchl may be determined for the LH 1 complex because its subunit can be studied by reconstitution under equilibrium conditions. Important to the determination and utilization of such information is the characterization of the interacting molecular species. To aid in this characterization, a fluorescent probe molecule has been covalently attached to each of the LH 1 polypeptides. The fluorescent probes were selected for optimal absorption and emission properties in order to facilitate their unique excitation and to enable the detection of energy transfer to Bchl. Oregon Green 488 carboxylic acid and 7-diethylaminocoumarin-3-carboxylic acid seemed to fulfill these requirements. Each of these probes were utilized to derivatize the LH1 β-polypeptide of Rhodobacter sphaeroides. It was demonstrated that the β-polypeptides did not interact with each other in the absence of Bchl. When Bchl was present, the probe-labeled β-polypeptides interacted with Bchl to form subunit-type complexes much as those formed with the native polypeptides. Energy transfer from the probe to Bchl occurred with a high efficiency. The α-polypeptide from LH 1 of Rb. sphaeroides and that from Rhodospirillum rubrum were also derivatized in the same manner. Since these polypeptides do not oligomerize in the absence of a β-polypeptide, reversible binding of a single Bchl to a single polypeptide could be measured. Dissociation constants for complex formation were estimated. The relevance of these data to earlier studies of equilibria involving subunit complexes is discussed. Also involved in the photoreceptor complex of Rb. sphaeroides and Rhodobacter capsulatus is another protein referred to as PufX. Two large segments of this protein were chemically synthesized, one reproducing the amino acid sequence of the core segment predicted for Rb. sphaeroides PufX and the other reproducing the amino acid sequence predicted for the core segment of Rb. capsulatus PufX. Each polypeptide was covalently labeled with a fluorescent probe and tested for energy transfer to Bchl. Each was found to bind Bchl with an affinity similar to the affinity of the LH 1 polypeptides for Bchl. It is suggested that PufX binds Bchl and interacts with a Bchlċα-polypeptide component of LH 1 to truncate, or interupt, the LH 1 ring adjacent to the location of the QB binding site of the RC.

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  1. Paul A. Loach

    Present address: Division of Biochemistry and Molecular Biology, University of Glasgow, Microbial Photosynthesis Lab, Room 217 Davidson Building, Glasgow, G128QQ, UK

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  1. Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Hogan Hall, Room 2-100, 2205 Tech Dr., Evanston, IL, 60208-3500, USA

    Christopher J. Law, Jennifer Chen, Pamela S. Parkes-Loach & Paul A. Loach

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Law, C.J., Chen, J., Parkes-Loach, P.S. et al. Interaction of bacteriochlorophyll with the LH1 and PufX polypeptides of photosynthetic bacteria: use of chemically synthesized analogs and covalently attached fluorescent probes. Photosynthesis Research 75, 193–210 (2003). https://doi.org/10.1023/A:1023982327748

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