The enzyme ferredoxin-NADP+ oxidoreductase (FNR) from Synechococcus sp. PCC 7002 has an extended structure comprising three domains (FNR-3D) (Schluchter, W. M., and Bryant, D. A. (1992) Biochemistry 31, 3092-3102). Phycobilisome (PBS) preparations from wild-type cells contained from 1.0 to 1.6 molecules of FNR-3D per PBS, with an average value of 1.3 FNR per PBS. A maximum of two FNR-3D molecules could be specifically bound to wild-type PBS via the N-terminal, CpcD-like domain of the enzyme when exogenous recombinant FNR-3D (rFNR-3D) was added. To localize the enzyme within the PBS, the interaction of PBS and their substructures with rFNR-3D was further investigated. The binding affinity of rFNR-3D for phycocyanin (PC) hexamers, which contained a 22-kDa proteolytic fragment derived from CpcG, the L RC27 linker polypeptide, was higher than its affinity for PC hexamers containing no linker protein. PBS from a cpcD3 mutant, which lacks the 9-kDa, PC-associated rod linker, incorporated up to six rFNR-3D molecules per PBS. PBS of a cpcC mutant, which has peripheral rods that contain single PC hexamers, also incorporated up to six rFNR-3D molecules per PBS. Direct competition binding experiments showed that PBS from the cpcD3 mutant bound more enzyme than PBS from the cpcC mutant. These observations support the hypothesis that the enzyme binds preferentially to the distal ends of the peripheral rods of the PBS. These data also show that the relative affinity order of the PC complexes for FNR-3D is as follows: (αPCβ PC)6-LR33 > (α PCβPC)6-LRC27 > (αPCβPC)6. The data suggest that, during the assembly of the PBS, FNR-3D could be displaced to the periphery according to its relative binding affinity for different PC subcomplexes. Thus, FNR-3D would not interfere with the light absorption and energy transfer properties of PC in the peripheral rods of the PBS. The implications of this localization of FNR within the PBS with respect to its function in cyanobacteria are discussed.
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