Structural and functional analysis of the reducing side of photosystem I

Structural analysis of the reducing side of photosystem I (PSI) has been carried out using chemical cross-linking and monospecific antibodies. Incubation of PSI isolated from barley (Hordeum vulgare L.) with the hydrophilic cross-linking agent N-ethyl-3-3-(dimethylamino) propyl]-carbodiimide leads to cross-linking of the PSI-D subunit with the PSI-E and PSI-H subunits. In the presence of ferredoxin, cross-linking results in the formation of cross-linked products composed of PSI-D, PSI-E and ferredoxin and in a block in steady state NADP⁺ photoreduction. No cross-linking of ferredoxin occurs at elevated ionic strength or using heat-denatured ferredoxin. Cross-linking of ferredoxin does not inhibit electron transfer from plastocyanin to methyl viologen. Steady state NADP⁺ photoreduction was analyzed in PSI or thyla-koids incubated with antibodies against individual PSI subunits. Incubation with antibodies against PSI-C, -H, -I, or -L had no effect on PSI activity, whereas antibodies against PSI-D or PSI-E had similar effects and caused a large decrease in activity. The results provide evidence that the PSI-D and PSI-E subunits are localized on the reducing side of PSI, forming a barrier between PSI-C and the stroma as well as a docking site for ferredoxin. The PSI-H subunit has an exposed, stromal domain but this does not appear to contribute to the ferredoxin docking.