Reconstitution of barley photosystem I with modified PSI-C allows identification of domains interacting with PSI-D and PSI-A/B.
The PSI-C subunit of photosystem I shows similarity to soluble 2[4Fe-4S] ferredoxins. Alignment analysis clearly shows that PSI-C contains an 8-residue internal loop and a 15-residue C-terminal extension that are absent in the ferredoxins. The remaining residues in PSI-C are likely to be folded in a way similar to the soluble 2[4Fe-4S] ferredoxins. Two modified PSI-C subunits lacking either the 8-residue loop or 10 residues of the C terminus were expressed in Escherichia coli and used to reconstitute a barley P700-FX core prepared to specifically lack PSI-C, PSI-D, and PSI-E. As shown by EPR spectroscopy, the modified proteins carry two [4Fe-4S] clusters with characteristics similar to those of native PSI-C. Western blot analysis of the reconstituted photosystem I complexes showed that the modified PSI-C proteins bind to the P700-FX core. Flash photolysis revealed that in photosystem I complexes reconstituted in the presence of PSI-D with the C-terminally deleted PSI-C, the FA/FB back-reaction was less efficiently restored than with wild-type PSI-C. The loop-deleted PSI-C was even less efficient. We attribute these differences to altered binding properties of the modified proteins. Comparison of reconstitutions performed in the presence and absence of PSI-D shows that the loop-deleted PSI-C is unable to bind without PSI-D, whereas the C-terminally deleted PSI-C binds only weakly with PSI-D. These results imply that the internal loop of PSI-C interacts with the PSI-A/B heterodimer and that the C terminus of PSI-C interacts with PSI-D.
Naver
H
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Department of Plant Biology, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK 1871 Frederiksberg C, Copenhagen, Denmark.
Scott
M P
MP
Golbeck
J H
JH
Møller
B L
BL
Scheller
H V
HV
D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D001467:Hordeum; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry; D010777:Photochemistry; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry*; D045331:Photosystem I Protein Complex; D010940:Plant Proteins / Q000737:chemistry*; D011994:Recombinant Proteins; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D010940:Plant Proteins; D011994:Recombinant Proteins; D009249:NADP