The structure of the predicted amino acid sequence in the FX domain of Photosystem 1 was studied by molecular modeling and a working hypothesis was developed for the functional interaction of PsaC with the core heterodimer. We propose that the intervening sequences between homologous cysteines in the FX cluster form two flexible loops and participate in the binding of PsaC, and that the arginine residues in the two surface-exposed loops may promote the interaction between the P700-FX core and the subunit. The model was tested experimentally; chemical modification of arginine residues in the P700-FX core using phenylglyoxal prevented reconstitution of the core with PsaC and PsaD after insertion of FeS clusters in vitro. Treatment of the P700-FX core with trypsin also prevented reconstitution of terminal electron transfer to FAFB, although neither treatments affected the electron transfer to FX as judged by flash kinetic spectrophotometry. Electron transfer in the P700-FAFB complex was not impaired by either phenylglyoxal or trypsin treatment indicating that the small subunit(s) protect the arginine residues that become chemically modified or cleaved. The data are consistent with the working model and point to additional experiments designed to identify the specific residues involved in the interaction between the P700-FX core and PsaC.

We have postulated that the orientation of PsaC on the photosystem I core involves electrostatic interactions between charged residues on the core binding site and the subunit [Rodday, S. M., Jun, S.-S., & Biggins, J. (1993) Photosynth. Res. 36, 1-9]. We, therefore, changed eight acidic residues on PsaC to arginine and examined the efficiency of the mutant subunits in the reconstitution of P700-Fx cores in vitro. Reconstitution of the cores by the mutant subunits was determined by analysis of the kinetics of recombination reactions between P700+ and reduced acceptors as measured optically. Restoration of complete forward electron transfer, indicative of efficient subunit binding, was estimated from the ca. 30 ms decay component in the flash transients. Slightly reduced levels of reconstitution were observed for the mutants D24R, E46R/D47R. D61R, and E72R. In contrast, mutants D9R, E27R, and D32R showed significantly lower efficiencies. The presence of the iron-sulfur centers, FA and FB, in these three mutant subunits was confirmed by low-temperature EPR spectroscopy indicating that the polypeptides had folded correctly. We conclude that the introduction of positively charged side chains at positions 9, 27, and 32 seriously disrupts PsaC binding. However, when the wild-type acidic residues in these positions were changed to alanine, only mutant D9A showed a reduced level of reconstitution, suggesting that this aspartate is the most important residue participating in the electrostatic interaction with the core. The results are discussed in relation to the photosystem I crystal structure and support an orientation of PsaC on the core such that center FB is proximal to Fx.