Identification of the Subunit which Binds Plastocyanin

Photosystem I (PSI) has been isolated from mesophyll chloroplasts of mature maize leaves. The isolated PSI (PSI-200) was used as starting material for preparing an antenna-depleted core (PSI-100). Both of these preparations appear to be quite analogous to PSI complexes isolated from other plant tissue sources, such as those from C3 plants, as judged from NADP photoreduction assays, immunoblotting, and the ability of the complexes to form a covalent crosslinked product with spinach plastocyanin. The study suggests that the PSI complex from a C4 plant is similar to that isolated from a C3 plant in that both contain the plastocyanin docking protein although the apparent molecular weight of these respective subunits differ slightly. PSI complexes isolated from various higher plant sources (summarized in refs. 6 and 18) contain numerous polypeptides. Purified PSI complexes from spinach have been shown to contain at least 12 polypeptides. The functions as well as the polypeptide sequences of many of these subunits have been elucidated in recent years. The primary reactants (P700, AO, Al, Fe-Sx; see refs. 7, 8, 14, 16, 29) are bound to two similar yet distinct 84 kD subunits (psaA and psaB), and these subunits also bind approximately 100 Chl a molecules (14, 29). The two Fe-S clusters (Fe-SA and Fe-SB) are bound to a single polypeptide of 9 kD (24, 30), which has been identified as the psaC gene product (11, 23). Three of the polypeptides in the 24 to 27 kD range have been identified as a light-harvesting antenna complex (LHCPI) which contains both Chl a and b and which can be removed from Pthe PSI complex by gentle detergent treatment (10, 16). This antenna complex has also been shown to bind approximately 100 Chl (10, 17). Recent experimental results from cross-linking studies suggest binding site functions for two additional polypeptides in the spinach PSI complex. A 22 kD subunit has been shown to interact with ferredoxin on the acceptor side of PSI (32, 33), while a 19 kD subunit has been shown to play a similar role on the donor side of PSI, with plastocyanin (31). The 19 kD subunit is removed from the PSI complex by the same detergent treatment which removes the LHCPI complex (31). This subunit was previously identified as subunit III by Bengis and Nelson (3). Both of these PSI polypeptides are ' Supported in part by a grant from the National Science Foundation (DMB-871 1206). nuclear-encoded and their sequences have recently been reported. The gene for the 22 kD subunit has referred to as psaD and sequences of four psaD gene products have been reported (5, 13, 16, 26) from higher plants. The N-terminal sequence for the 19 kD subunit from spinach (our unpublished data) matches the gene which has recently been cloned (27) and designated as psaF. This gene is similar to psaD in that the polypeptide is basic and contains highly charged amino acids throughout the N-terminal region of the mature polypeptide. It has been proposed that this subunit is part of the binding site for plastocyanin (3, 9) and enhances rapid electron transfer to P700 (25). The only direct evidence for the association of this subunit with plastocyanin has come from cross-linking studies with the water soluble carbodiimide crosslinker, EDC2 (31). One anomaly in this situation is the report of Nechushtai et al. (21, 22) that a PSI complex from the C4 plant, maize, does not contain subunit III, the 19 kD subunit which interacts with plastocyanin. In contrast, Ratajczak et al. (25) have shown that plastocyanin is as effective a donor to maize PSI as it is to the PSI complex from a C3 plant, such as spinach. Because of our continuing interest in the interaction of plastocyanin with the donor side of PSI, we have characterized the PSI complex from a C4 plant and compared it with an analogous complex from a C3 plant. The interaction ofplastocyanin with maize PSI has been examined by utilizing NADP photoreduction assays, immunoblotting, and EDC cross-linking, and these methods have demonstrated unambiguously the presence of a plastocyanin binding subunit in maize PSI. These results suggest that PSI complexes from C3 and C4 plant PSI complexes have a strong functional and structural similarity. MATERIALS AND METHODS