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A series of deletions from the carboxyl terminus of the 23-kD subunit of the photosynthetic oxygen-evolving complex OE23 revealed that these truncations result i n various degrees of inhibition of translocation across thylakoid membranes and their subsequent assembly to the oxygen-evolving complex. lmport of in vitro translated precursors across the chloroplast envelopes was not inhibited by these truncations. Time-course studies of the import of truncated OE23 precursors into intact chloroplasts revealed that the stromal intermediate was subsequently translocated into the thylakoid lumen, where it was processed to a smaller size and rapidly degraded.I n contrast to the full-length OE23 intermediate, the truncated intermediate forms that accumulated in the stroma as a result of de-energization of thylakoid membranes could be found associated with the membrane rather than free in the stroma. Protease digestion experiments revealed that the deletions evidently altered the folded conformation of the protein. These results suggest that the carboxyl-terminal portion of the OE23 precursor is important for the maintenance of an optimal structure for import into thylakoids, implying that the efficient translocation of OE23 requires the protein to be correctly folded. In addition, the rapid degradation of the truncated forms of the processed OE23 within the lumen indicates that a protease (or proteases) active in the lumen can recognize and remove misfolded polypeptides.Within the chloroplast, the multisubunit PSII protein complex is the site of catalysis for the conversion of water to molecular oxygen as a result of photosynthetic electron transfer across thylakoid membranes. The minimal PSII preparation from eukaryotic photosynthetic membranes that is able to catalyze oxygen evolution consists of the 32-and 34-kD integral protein subunits known as D1 and D2, which make up the core of the reaction center; the a and p subunits of Cyt b559; the core antenna components CP43 and CP47; and severa1 other small polypeptides (reviewed by Debus, 1992;Vermaas, 1993). Most oxygen-evolving preparations also contain a 33-kD extrinsic protein (OE33), although its presence is not strictly required for this activity. Two additional extrinsic protein components of 23 and 17 kD (OE23 and OE17) also are associated with a more intact oxygen-evolving membrane preparation. The extrinsic subunits of the OEC, OE33,OE23, and OE17 are tightly bound to the lumen-exposed domains of the PSII complex. Loss of the OE23 and OE17 proteins has been correlated with lowered rates of oxygen evolution, but because depleted membranes retain the ability to catalyze water oxidation, these polypeptides are considered to have structural and regulatory roles, as opposed to catalytic functions, in oxygen evolution (Vermaas, 1993).The assembly of the OEC is of interest not only for its implications in mechanistic and regulatory aspects of oxygen evolution, but also because the multimeric PSII-OEC is formed with subunits encoded by both chloroplast and nuclear genes...

Using a cotransformation system to identify chloroplast transformants in Chlamydomonas reinhardt&, we converted histidine-195 of the photosystem H reaction center D1 protein to a tyrosine residue. The mutants were characterized by a reduced quantum efficiency for photosynthetic oxygen evolution, which varied in a pH-dependent manner, a reduced capacity to oxidize artificial donors to photosystem H, and P680) reduction kinetics (microsecond) that were essentially similar to wild type. In addition, a dark-stable radical was detected by ESR in mutant photosystem II particles but not in wild-type particles. This radical was similar in g value and lineshape to chlorophyll or carotenoid cations but could have arisen from a tyrosine-195 cation. The ability of the photosystem II trap (P680) to oxidize tyrosine residues suggests that the mutant tyrosine residue could be used as a redox-sensitive probe to investigate the environment around the photosystem H trap.It is generally accepted that the photosystem II (PSII) reaction center of chloroplasts is structurally and functionally similar to the bacterial photosynthetic reaction center (1-5). Both reaction center types have two core polypeptides that coordinate the primary electron donor, presumably a chlorophyll (Chl) special pair, and the primary electron acceptors, pheophytin and the plastoquinone Qa. These polypeptides, the L and M subunit of the bacterial reaction center and the D1 and D2 subunits of PSII, have significant levels of sequence identity (25%) (6). In addition, the D1 and D2 polypeptides have the same number and orientations of transmembrane domains as the L and M subunits (3). These and other similarities suggest that the protein domains involved in the coordination of the PSII trap and binding of electron acceptors are structurally similar to those of the L and M polypeptides of the bacterial reaction center (1-3). The bacterial and PSII reaction centers, however, differ with respect to the organization of secondary electron donors and the redox potential of the reaction center traps. The Chl special pair of the bacterial reaction center is reduced by a cytochrome, whereas the PSII trap, P680', is reduced by a peptidyl tyrosine residue that in turn is reduced by electrons derived from the oxidation ofwater. Since the redox potential of a Chl dimer in solution is not sufficiently positive to drive the oxidation of water, it has been generally assumed that the redox potential of the PSII trap is determined by interactions between the chromophores and proteins. Modified Chl molecules containing point charges or the presence of charged residues near Chl may alter spectral properties or the redox potential of Chl (7-9). A survey of the amino acid sequences of the PSII core polypeptides D1 and D2 was conducted to identify conserved and potentially charged residues that could influence the electronic properties of the PSII trap. We identified a potentially ionizable residue, histidine-195 (H195) of the D1 protein, which is projected to be adjacent to ...