On the lumenal side of photosystem I (PSI), each of the two large core subunits, PsaA and PsaB, expose a conserved tryptophan residue to the surface. PsaB-Trp627is part of the hydrophobic recognition site that is essential for tight binding of the two electron donors plastocyanin and cytochromec6to the donor side of PSI (Sommer, F., Drepper, F., and Hippler, M. (2002)J. Biol. Chem.277, 6573–6581). To examine the function of PsaA-Trp651in binding and electron transfer of both donors to PSI, we generated the mutants PsaA-W651F and PsaA-W651S by site-directed mutagenesis and biolistic transformation ofChlamydomonas reinhardtii.The protein-protein interaction and the electron transfer between the donors and PSI isolated from the mutants were analyzed by flash absorption spectroscopy. The mutation PsaA-W651F completely abolished the formation of a first order electron transfer complex between plastocyanin (pc) and the altered PSI and increased the dissociation constant for binding of cytochrome (cyt)c6by more than a factor of 10 as compared with wild type. Mutation of PsaA-Trp651to Ser had an even larger impact on the dissociation constant. TheKDvalue increased another 2-fold when the values obtained for the interaction and electron transfer between cytc6and PSI from PsaA-W651S and PsaA-W651F are compared. In contrast, binding and electron transfer of pc to PSI from PsaA-W651S improved as compared with PSI from PsaA-W651F and admitted the formation of an inter-molecular electron transfer complex, resulting in aKDvalue of about 554 μmthat is still five times higher than observed for wild type. These results demonstrate that PsaA-Trp651is, such as PsaB-Trp627, crucial for high affinity binding of pc and cytc6to PSI. Our results also indicate that the highly conserved structural recognition motif that is formed by PsaA-Trp651and PsaB-Trp627confers a differential selectivity in binding of both donors to PSI.
Cloning and Sequencing of a Full-Length cDNA Clone Encoding the PSI-D Subunit of Photosystem I from Barley