The Eight-amino Acid Internal Loop of PSI-C Mediates Association of Low Molecular Mass Iron-Sulfur Proteins with the P700-FXCore in Photosystem I

Helle Naver,M Paul Scott,John H Golbeck,Carl Erik Olsen,Henrik V Scheller

doi:10.1074/jbc.273.30.18778

Helle Naver, M Paul Scott + Show 3 more

Open Access

https://doi.org/10.1074/jbc.273.30.18778

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Abstract

The PSI-C subunit of photosystem I (PS I) shows similarity to soluble 2[4Fe-4S] ferredoxins. PSI-C contains an eight residue internal loop and a 15 residue C-terminal extension which are absent in the ferredoxins. The eight-residue loop has been shown to interact with PSI-A/PSI-B (Naver, H., Scott, M. P., Golbeck, J. H., Moller, B. L., and Scheller, H. V. (1996) J. Biol. Chem. 271, 8996-9001). Four mutant proteins were constructed. Two were modified barley PSI-C proteins, one lacking the loop and the C terminus (PSI-Ccore) and one where the loop replace the C-terminal extension (PSI-CcoreLc-term). Two were modified Clostridium pasteurianum ferredoxins, one with the loop of barley PSI-C and one with both the loop and the C terminus of PSI-C. Wild-type proteins and the mutants were used to reconstitute barley P700-FX cores lacking PSI-C, -D, and-E. Western blotting showed that PSI-CcoreLc-term binds to PS I, whereas PSI-Ccore does not. Without PSI-D the PSI-CcoreLc-term mutant accepts electrons from FX in contrast to PSI-C mutants without the loop. Flash photolysis of P700-FX cores reconstituted with C. pasteurianum ferredoxin showed that only the ferredoxin mutants with the loop accepted electrons from FX. From this, it is concluded that the loop of PSI-C is necessary and sufficient for the association between PS I and PSI-C, and that the loop is functional as an interaction domain even when positioned at the C terminus of PSI-C or on a low molecular mass, soluble ferredoxin.

Highlights

The PSI-C subunit of photosystem I (PS I) shows similarity to soluble 2[4Fe– 4S] ferredoxins
It is concluded that the loop of PSI-C is necessary and sufficient for the association between PS I and PSI-C, and that the loop is functional as an interaction domain even when positioned at the C terminus of PSI-C or on a low molecular mass, soluble ferredoxin
We reported that the eight-residue loop and the C-terminal addition play a role in the interaction of PSI-C with PSI-A/B and PSI-D, respectively [18]

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction and Protein Isolation—The production of the PSI-C expression clone from barley, PSI-C⌬L and PSI-C⌬C has been described previously [8, 18]. To produce barley PSI-C lacking both the loop and the C-terminal domain, appropriate oligonucleotides were used as primers in a polymerase chain reaction with psaC⌬L as template to yield one fragment. To produce the C. pasteurianum ferredoxin (Cl.Fd) with the loop inserted, four oligonucleotides containing the loop from PSI-C, were used with the Cl.Fd gene in two polymerase chain reactions to yield the two products: the 5Ј end of the gene with the whole loop and the 3Ј end with the whole loop. NADPϩ photoreduction measurements of the samples containing the clostridial ferredoxin proteins were carried out under strictly anaerobic conditions maintained using the glucose oxidase/catalase system as described by Kjaer and Scheller [35]. Western blot analysis was carried out by transferring electrophoresed proteins to nitrocellulose membranes followed by incubation with monospecific rabbit antibodies [32] and visualized using a secondary antibody conjugated with alkaline phosphatase (DAKO, Copenhagen, Denmark)

RESULTS

64 Ϯ 5 85 Ϯ 3 12 Ϯ 10 60 Ϯ 3

DISCUSSION

13 Ϯ 3 73 Ϯ 2 84 Ϯ 4