We describe the three-dimensional structure of higher plant photosystem I (PSI) as obtained by electron microscopy of two-dimensional crystals formed at the grana margins of thylakoid membranes. The negatively stained crystalline areas displayed unit cell dimensions a ‫؍‬ 26.6 nm, b ‫؍‬ 27.7 nm, and ␥ ‫؍‬ 90 o , and p22 1 2 1 plane group symmetry consisting of two monomers facing upward and two monomers facing downward with respect to the membrane plane. Higher plant PSI shows several structural similarities to the cyanobacterial PSI complex, with a prominent ridge on the stromal side of the complex. The stromal ridge is resolved into at least three separate domains that are interpreted as representing the three well characterized stromal subunits, the psa C, D, and E gene products. The lumenal surface is relatively flat but exhibits a distinct central depression that may be the binding site for plastocyanin. Higher plant PSI is of dimensions 15-16 ؋ 11-12.5 nm, and thus leaves a larger footprint in the membrane than its cyanobacterial equivalent (13 ؋ 10.5 nm). It is expected that additional membrane-bound polypeptides will be present in the higher plant PSI. Both higher plant and cyanobacterial complexes span about 8 -9 nm in the direction orthogonal to the membrane. This report represents the first three-dimensional structure for the higher plant PSI complex.Photosystem (PS) 1 I is a multiprotein complex found in the photosynthetic membranes of plants and cyanobacteria. It carries out the second light-driven electron transfer step in the linear Z scheme of photosynthesis (1). In higher plants, a significant proportion of the PSI complexes is located in the stromal lamellae (2). Cyclic electron transfer may be carried out by this pool of PSI for at least part of the time. It is also thought that higher plant PSI complexes may be located around the periphery of the grana stacks (the grana margins) where they interact with photosystem II (PSII) complexes, and probably take part in linear electron transfer (2). On the basis of spectroscopic data, it has been proposed that there is heterogeneity in the structure of higher plant PSI (3), with stromally located PSI complexes predicted to have a smaller photosynthetic unit size (i.e. a reduced number of light-harvesting pigments) than their granally located sisters. Until now, there has been little direct structural investigation of higher plant PSI. The paucity of structural data was largely due to the lack of two-or threedimensional crystals of the higher plant complex. In the absence of crystals, the analysis of freeze-fractured or freezeetched thylakoid membranes by shadowing and electron microscopy was carried out (e.g. see Dunahay and Staehelin (4), but only the overall dimensions of the complexes were determined, and uncertainty over their identification as PSI was not eliminated. In a second approach, higher plant PSI complexes have been purified after detergent solubilization and then examined by electron microscopy after negative staining (5). These studies...

In this study, we present the location of the ferredoxin-binding site in photosystem I from spinach. Image analysis of negatively stained two-dimensional crystals indicates that the addition of ferredoxin and chemical cross-linkers do not significantly alter the unit cell parameters (for untreated photosystem I, a ‫؍‬ 26.4 nm, b ‫؍‬ 27.6 nm, and ␥ ‫؍‬ 90°, space group p22 1 2 1 and for ferredoxin cross-linked photosystem I, a ‫؍‬ 26.2 nm, b ‫؍‬ 27.2 nm, and ␥ ‫؍‬ 90°, space group p22 1 2 1 ). Fourier difference analysis reveals that ferredoxin is bound on top of the stromal ridge principally interacting with the extrinsic subunits PsaC and PsaE. This location would be accessible to the stroma, thereby promoting efficient electron transfer away from photosystem I. This observation is significantly different from that of the ferredoxin binding site proposed for cyanobacteria. A model for the binding of ferredoxin in vascular plants is proposed and is discussed relative to observations in cyanobacteria.The light reactions of oxygenic photosynthesis are found in specialized membranes and are catalyzed by pigment protein complexes. PS-I 1 is located at the edges of the granal stacks and in the stromal lamellae in plants (1, 2). It is that part of the light reactions of photosynthesis that is responsible for the absorption of light energy and the generation of reduced Fd via a series of electron carriers across the thylakoid membrane. The mobile electron carrier Fd has been shown to be involved in a wide variety of redox reactions in plants. These include mediating the transfer between PS-I and NADP ϩ in linear electron transfer and playing a key role in cyclic photophosphorylation (reviewed in Refs. 3 and 4). Fd also plays a significant role in the redox reactions of glutamate synthase, sulfate reductase, nitrite reductase, and Fd-thioredoxin oxidoreductase (5). The interaction of Fd with PS

Crystallization trials using three polyoxyethylene surfactants as precipitating agents are described. Of the eight soluble proteins screened, five were successfully crystallized at the first attempt. These included lysozyme, catalase, ferritin, ribonuclease A and ubiquitin. Further work suggested that these surfactants could also be suitable for cryo-crystallographic analysis of crystals. At the concentrations used in the crystallization trials [10-40%(v/v)], they are capable of promoting the formation of non-crystalline glasses at cryogenic temperatures (77 K). This would facilitate crystal mounting and allow the minimization of crystal irradiation damage. Results from this study also suggest that proteins remain stable at high concentrations of these surfactants [40%(w/v)] and over long time periods (>1 month). A number of membrane proteins were also screened for crystallization. These included photosystems I and II and light harvesting complexes I and II from spinach and bacteriorhodopsin from Halobacterium halobium. The trials were unsuccessful both in the absence and presence of heptane-l,2,3-triol and over a wide range of surfactant concentrations.

We have studied the binding sites of the electron donor and acceptor proteins of vascular plant photosystem I by electron microscopy/crystallography. Previously, we identified the binding site for the electron acceptor (ferredoxin). In this paper we complete these studies with the characterization of the electron donor (plastocyanin) binding site. After cross-linking, plastocyanin is detected using Fourier difference analysis of two dimensionally ordered arrays of photosystem I located at the periphery of chloroplast grana. Plastocyanin binds in a small cavity on the lumenal surface of photosystem I, close to the center and with a slight bias toward the PsaL subunit of the complex. The recent release of the full coordinates for the cyanobacterial photosystem I reaction center has allowed a detailed comparison between the structures of the eukaryotic and prokaryotic systems. This reveals a very close homology, which is particularly striking for the lumenal side of photosystem I.The multisubunit protein complexes responsible for the light reactions of photosynthesis are found in the thylakoid membranes within the chloroplasts of vascular plants. Photosystem I (PS-I) 1 is found at the edges of the granal stacks and in the stromal lamellae of these thylakoid membranes (1, 2). PS-I catalyzes the part of the light reactions responsible for the absorption of light energy and generating reduced ferredoxin via a series of electron carriers across the thylakoid membrane. Following charge separation in PS-I, the oxidized reaction center chlorophyll pair (P700 ϩ ) is reduced by plastocyanin (Pc). The oxidized Pc is ultimately reduced in the cytochrome b 6 f complex (reviewed in Ref.3).The three-dimensional structure of a cyanobacterial PS-I reaction center has been described for Synechococcus elongatus (4 -8) to a current resolution better than 3 Å resolution. This structure does not show the location of the mobile electron carriers Pc and ferredoxin (Fd) as they are lost during the purification of the complex.Two-dimensional crystals of a P22 1 2 1 plane group occurring at the edges of grana in spinach thylakoids have been shown to contain PS-I and have yielded structural data to 2.7 nm (2) and refined three-dimensional data to 2.5 nm resolution (9). Unlike cyanobacteria, spinach PS-I does not appear to form trimers but does show many common structural features (2, 9). In neither of the studies on spinach (2, 9) were the mobile electron carriers Pc and Fd retained. However, studies on the binding site for Fd, the terminal mobile electron acceptor in PS-I, have recently been possible (10) using a cross-linking and image analysis approach.In-vivo, cyanobacterial and algal PS-I can be reduced by either Pc or the alternative electron donor cytochrome c6, whereas vascular plant PS-I is reduced only by Pc. Structural insight into Pc binding in cyanobacteria has been derived from modeling studies using PS-I from the cyanobacterium S. elongatus and Pc from Enteromorpha prolifera (a green alga) (7). This study concluded that the li...

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