Ferredoxin:NADP oxidoreductases (FNRs) constitute a family of flavoenzymes that catalyze the exchange of reducing equivalents between one-electron carriers and the two-electron-carrying NADP(H). The main role of FNRs in cyanobacteria and leaf plastids is to provide the NADPH for photoautotrophic metabolism. In root plastids, a distinct FNR isoform is found that has been postulated to function in the opposite direction, providing electrons for nitrogen assimilation at the expense of NADPH generated by heterotrophic metabolism. A multiple gene family encodes FNR isoenzymes in plants, whereas there is only one FNR gene (petH) in cyanobacteria. Nevertheless, we detected two FNR isoforms in the cyanobacterium Synechocystis sp. strain PCC6803. One of them (FNR S Ϸ34 kDa) is similar in size to the plastid FNR and specifically accumulates under heterotrophic conditions, whereas the other one (FNRL Ϸ46 kDa) contains an extra N-terminal domain that allows its association with the phycobilisome. Site-directed mutants allowed us to conclude that the smaller isoform, FNR S, is produced from an internal ribosome entry site within the petH ORF. Thus we have uncovered a mechanism by which two isoforms are produced from a single gene, which is, to our knowledge, novel in photosynthetic bacteria. Our results strongly suggest that FNRL is an NADP ؉ reductase, whereas FNRS is an NADPH oxidase.

Of the stroma‐accessible proteins of photosystem I (PSI) from Synechocystis sp. PCC 6803, the PSI‐C, PSI‐D and PSI‐E subunits have already been characterized, and the corresponding genes isolated. PCR amplification and cassette mutagenesis were used in this work to delete the psaE gene. PSI particles were isolated from this mutant, which lacks subunit PSI‐E, and the direct photoreduction of ferredoxin was investigated by flash absorption spectroscopy. The second order rate constant for reduction of ferredoxin by wild type PSI was estimated to be approximately 10(9) M‐1s‐1. Relative to the wild type, PSI lacking PSI‐E exhibited a rate of ferredoxin reduction decreased by a factor of at least 25. After reassociation of the purified PSI‐E polypeptide, the original rate of electron transfer was recovered. When a similar reconstitution was performed with a PSI‐E polypeptide from spinach, an intermediate rate of reduction was observed. Membrane labeling of the native PSI with fluorescein isothiocyanate allowed the isolation of a fluorescent PSI‐E subunit. Peptide analysis showed that some residues following the N‐terminal sequence were labeled and thus probably accessible to the stroma, whereas both N‐ and C‐terminal ends were probably buried in the photosystem I complex. Site‐directed mutagenesis based on these observations confirmed that important changes in either of the two terminal sequences of the polypeptide impaired its correct integration in PSI, leading to phenotypes identical to the deleted mutant. Less drastic modifications in the predicted stroma exposed sequences did not impair PSI‐E integration, and the ferredoxin photoreduction was not significantly affected. All these results lead us to propose a structural role for PSI‐E in the correct organization of the site involved in ferredoxin photoreduction.

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The reaction center of photosystem I (PSI) reduces soluble ferredoxin on the stromal side of the photosynthetic membranes of cyanobacteria and chloroplasts. The X-ray structure of PSI from the cyanobacterium Synechococcus elongatus has been recently established at a 2.5 A resolution [Nature 411 (2001) 909]. The kinetics of ferredoxin photoreduction has been studied in recent years in many mutants of the stromal subunits PsaC, PsaD and PsaE of PSI. We discuss the ferredoxin docking site of PSI using the X-ray structure and the effects brought by the PSI mutations to the ferredoxin affinity.