Insertion of the N-terminal part of PsaF from Chlamydomonas reinhardtii into photosystem I from Synechococcus elongatus enables efficient binding of algal plastocyanin and cytochrome c6

Abstract

A strain of the cyanobacterium Synechococcus 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. reinhardtii that 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 X 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 from S. elongatus and the algal donors. At high concentrations of C. reinhardtii cytochrome c6, a fast first-order kinetic component (t 1/2 = 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. Crosslinking studies show that, in contrast to PSI from wild type S. 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.