A strain of the cyanobacteriumSynechococcus elongatus was generated that expresses a hybrid version of the photosystem I subunit PsaF consisting of the first 83 amino acids of PsaF from the green alga Chlamydomonas reinhardtii fused to the C-terminal portion of PsaF from S. elongatus. The corresponding modified gene was introduced into the genome of the psaF-deletion strain FK2 by cointegration with an antibiotic resistance gene. The transformants express a new PsaF subunit similar in size to PsaF from C. reinhardtiithat is assembled into photosystem I (PSI). Hybrid PSI complexes isolated from these strains show an increase by 2 or 3 orders of magnitude in the rate of P700⁺ reduction by C. reinhardtii cytochrome c₆ or plastocyanin in 30% of the complexes as compared with wild type cyanobacterial PSI. The corresponding optimum second-order rate constants (k₂ = 4.0 and 1.7 × 10⁷ m¹ s¹ for cytochrome c₆ and plastocyanin) are similar to those of PSI from C. reinhardtii. The remaining complexes are reduced at a slow rate similar to that observed with wild type PSI fromS. elongatus and the algal donors. At high concentrations of C. reinhardtii cytochrome c₆, a fast first-order kinetic component (t1/2 = 4 μs) is revealed, indicative of intramolecular electron transfer within a complex between the hybrid PSI and cytochrome c₆. This first-order phase is characteristic for P700⁺ reduction by cytochrome c₆ or plastocyanin in algae and higher plants. However, a similar fast phase is not detected for plastocyanin. Cross-linking studies show that, in contrast to PSI from wild typeS. elongatus, the chimeric PsaF of PSI from the transformed strain cross-links to cytochrome c₆ or plastocyanin with a similar efficiency as PsaF from C. reinhardtii PSI. Our data indicate that development of a eukaryotic type of reaction mechanism for binding and electron transfer between PSI and its electron donors required structural changes in both PSI and cytochrome c₆ or plastocyanin.