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Photosystem I (PSI) interacts with plastocyanin or cytochrome c 6 on the luminal side. To identify sites of interaction between plastocyanin/cytochrome c 6 and the PSI core, site-directed mutations were generated in the luminal J loop of the PsaB protein from Synechocystis sp. PCC 6803. The eight mutant strains differed in their photoautotrophic growth. Western blotting with subunit-specific antibodies indicated that the mutations affected the PSI level in the thylakoid membranes. PSI proteins could not be detected in the S600R/G601C/ N602I, N609K/S610C/T611I, and M614I/G615C/W616A mutant membranes. The other mutant strains contained different levels of PSI proteins. Among the mutant strains that contained PSI proteins, the H595C/L596I, Q627H/L628C/I629S, and N638C/N639S mutants showed similar levels of PSI-mediated electron transfer activity when either cytochrome c 6 or an artificial electron donor was used. In contrast, cytochrome c 6 could not function as an electron donor to the W622C/A623R mutant, even though the PSI activity mediated by an artificial electron donor was detected in this mutant. Thus, the W622C/A623R mutation affected the interaction of the PSI complex with cytochrome c 6 . Biotin-maleimide modification of the mutant PSI complexes indicated that His-595, Trp-622, Leu-628, Tyr-632, and Asn-638 in wildtype PsaB may be exposed on the surface of the PSI complex. The results presented here demonstrate the role of an extramembrane loop of a PSI core protein in the interaction with soluble electron donor proteins.Photosystem I (PSI) 1 is a multisubunit membrane-protein complex that catalyzes electron transfer from the reduced plastocyanin in the thylakoid lumen to the oxidized ferredoxin in the chloroplast stroma or cyanobacterial cytoplasm (1-4). In cyanobacteria and green algae, cytochrome c 6 can substitute plastocyanin depending on the growth conditions (5-7). The crystal structure of PSI at 4-Å resolution is available (8, 9). PSI contains 89 chlorophyll a molecules, 83 of which constitute the core antenna system. The PsaA and PsaB subunits form the heterodimeric core that harbors most of the antenna chlorophyll a molecules, ␤-carotenes, the primary electron donor P700, and a chain of electron acceptors (A 0 , A 1 , and F X ). In addition to the core proteins, the cyanobacterial PSI complex contains three peripheral proteins (PsaC, PsaD, and PsaE) and six integral membrane proteins (PsaF, PsaI, PsaJ, PsaK, PsaL, and PsaM) (1-3). The PsaC, PsaD, and PsaE subunits constitute the reducing side of the PSI complex. PsaC binds the terminal electron acceptors F A and F B , which donate electrons to ferredoxin. PsaD and PsaE facilitate the docking of ferredoxin on PSI (1). Functions of the other subunits have been studied by using subunit-deficient mutants of cyanobacteria and algae (10 -17).On the luminal side of thylakoid membranes, PSI accepts electrons from plastocyanin or cytochrome c 6 . The interaction of plastocyanin with plant and algal PSI complexes shows a fast kinetic step, which can be at...

Wild-type plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803 does not form any kinetically detectable transient complex with Photosystem I (PS I) during electron transfer, but the D44R/D47R double mutant of copper protein does [De la Cerda et al. (1997) Biochemistry 36: 10125-10130]. To identify the PS I component that is involved in the complex formation with the D44R/D47R plastocyanin, the kinetic efficiency of several PS I mutants, including a PsaF-PsaJ-less PS I and deletion mutants in the lumenal H and J loops of PsaB, were analyzed by laser flash absorption spectroscopy. The experimental data herein suggest that some of the negative charges at the H loop of PsaB are involved in electrostatic repulsions with mutant plastocyanin. Mutations in the J loop demonstrate that this region of PsaB is also critical. The interaction site of PS I is thus not as defined as first expected but much broader, thereby revealing how complex the evolution of intermolecular electron transfer mechanisms in photosynthesis has been.

PsaA and PsaB are homologous integral membrane proteins that form the heterodimeric core of photosystem I. Domain-specific antibodies were generated to examine the topography of PsaA and PsaB. The purified photosystem I complexes from the wild type strain of Synechocystis sp. PCC 6803 were treated with eight proteases to study the accessibility of cleavage sites in PsaA and PsaB. Proteolytic fragments were identified using the information from N-terminal amino acid sequencing, reactivity to antibodies, apparent mass, and specificity of proteases. The extramembrane loops of PsaA and PsaB differed in their accessibility to proteases, which indicated the folded structure of the loops or their shielding by the small subunits of photosystem I. NaI-treated and mutant photosystem I complexes were used to identify the extramembrane loops that were exposed in the absence of specific small subunits. The absence of PsaD exposed additional proteolytic sites in PsaB, whereas the absence of PsaE exposed sites in PsaA. These studies distinguish PsaA and PsaB in the structural model for photosystem I that has been proposed on the basis of x-ray diffraction studies (Krauß, N.,

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