Surface analysis of the photosystem I complex by electron and atomic force microscopy¹

Abstract

Two-dimensional (2D) crystals of the photosystem I (PSI) reaction center from Synechococcus sp. OD24 were analyzed by electron and atomic force microscopy. Surface relief reconstructions from electron micrographs of freeze-dried unidirectionally shadowed samples and topographs recorded with the atomic force microscope (AFM) provided a precise definition of the lumenal and stromal PSI surfaces. The lumenal surface was composed of four protrusions that surrounded an indentation. One of the protrusions, the PsaF subunit, was often missing. Removal of the extrinsic proteins with the AFM stylus exposed the stromal side of the PSI core, whose surface structure could then be imaged at a resolution better than 1.4 nm. This interfacial surface between core and extrinsic subunits, had a pseudo-2-fold symmetry and protrusions that correlated with the surface helices e and e′ or were at the sites of putative α-helix-connecting loops estimated from the 4 Å map of the complex. The molecular dissection achieved with the AFM, opens new possibilities to unveil the interfaces between subunits of supramolecular assemblies.

Introduction

Photosystem I (PSI) is one of the two pigment-containing reaction centers of oxygenic photosynthesis found in cyanobacteria and plants (Barber & Andersson, 1994). It catalyzes the light-dependent transfer of electrons from reduced plastocyanin or cytochrome c₆ to soluble ferredoxin or flavodoxin across the thylakoid membrane. The functional reaction center of the thermophilic cyanobacterium Synechococcus sp. consists of 11 protein subunits and 90 chlorophyll molecules, assembled into a 340 kDa complex Krauss et al 1993, Golbeck 1994. The two high molecular mass subunits PsaA and PsaB, 83 kDa each, are very hydrophobic and bind the electron transfer components P700, A₀, A₁, F_X, an unspecified number of β-carotene molecules and most of the chlorophylls. The other nine subunits (PsaC, -D, -E, -F, -I, -J, -K, -L and -M) are small, each having a mass below 20 kDa.

The extrinsic protein PsaC (9 kDa) at the stromal side contains two [4Fe-4S]-clusters, the F_Aand F_Bcenters, which are the terminal electron acceptors in the electron transfer chain (Oh-oka et al., 1987). Thus, the electron released by photo-oxidation of P700 at the lumenal side tunnels through the cascade A₀, A₁, F_Xto reach F_A, F_Bin about 500 ns (Brettel, 1997). PsaC appears to bind loosely to the PsaA/PsaB heterodimer in the absence of the other two extrinsic proteins, PsaD (16 kDa) and PsaE (8 kDa). A stable binding of PsaC to the PsaA/PsaB core requires the supplementary binding of PsaD to the complex Zhao et al 1990, Li et al 1991. PsaD is also involved in the interaction between PSI and soluble ferredoxin during its photoreduction (Lelong et al., 1994), while PsaE is important for the cyclic electron transport (Zhao et al., 1993), as well as for the interaction between the terminal electron acceptor and ferredoxin Rousseau et al 1993, Sonoike et al 1993.

The function of the PsaF protein (15 kDa) at the lumenal side has been subject to discussion. In intact cells of the green alga Chlamydomonas reinhardtii, PsaF is implicated in the electron transfer from plastocyanin to oxidized P700 by providing a docking site for the electron donor: psaF⁻ mutants of this organism had a dramatically reduced electron transfer rate (Farah et al., 1995). In contrast, a psaF⁻ mutant of the cyanobacterium Synechocystis PCC 6803 exhibited normal electron transfer to P700⁺, implying that PsaF is not essential for the docking of either cytochrome c₆ or plastocyanin to PSI Xu et al 1994, Hippler et al 1996. While PSI is extracted as a mixture of trimers and monomers from thylakoid membranes of wild-type cyanobacteria, PSI from mutants that lack the PsaL protein (16 kDa) exists exclusively as a monomer after membrane solubilization (Chitnis & Chitnis, 1993). In addition, proteolysis studies have shown PsaL to be located about the 3-fold axis of the trimer, thus holding it together (Chitnis & Chitnis, 1993). Little is known about the function of the four other membrane intrinsic subunits (PsaI, -J, -K and -M) that have molecular masses ranging from 3 to 8 kDa (Golbeck, 1994).

A wealth of information about the PSI complex is available from different structure determination methods. Structural analysis of solubilized PSI by electron microscopy (EM) has provided evidence for the presence of trimeric complexes in the native membrane Hladik and Sofrova 1991, Tsiotis et al 1995. Analysis of solubilized trimeric complexes has allowed the positions of the subunits PsaC, -D, -E, -F and -J (Kruip et al., 1997), as well as the docking sites for ferredoxin (Lelong et al., 1996) and flavodoxin (Mühlenhoff et al., 1996) to be mapped. A three-dimensional reconstruction of negatively stained 2D crystals and a projection map of frozen-hydrated 2D crystals at 8 Å resolution (Karrasch et al., 1996), have given precise information on the integration of the PSI complex in the bilayer. The three-dimensional structure of PSI from the thermophilic cyanobacterium Synechococcus elongatus determined by X-ray crystallography is now available at a resolution of 4 Å (Schubert et al., 1997). This model reveals 34 transmembrane and nine surface α-helices, as well as the three [4Fe-4S] centers and 89 chlorophyll molecules per monomer.

Nevertheless, detailed structural information on the surface topography of this fascinating enzyme is lacking. This has prompted a study of PSI from Synechocystis sp. PCC 6803 using domain-specific antibodies and proteolysis (Sun et al., 1997). More direct approaches to investigate the surface structure of proteins are provided by microscopy. Surface relief reconstructions from electron micrographs of unidirectionally shadowed 2D crystals have yielded information to a resolution better than 1 nm (Walz et al., 1996). Topographs of membrane proteins in aqueous environment have been acquired at a resolution better than 1 nm using the atomic force microscope (AFM; Karrasch et al 1994, Schabert et al 1995, Muller et al 1995a) allowing single protein loops to be localized. The ability of the AFM to directly visualize protein conformational changes at high-resolution has also been demonstrated Muller et al 1995b, Muller et al 1996.

Here, we present the lumenal and stromal topography of the PSI complex when reconstituted in a lipid bilayer. The structural information of the native PSI reaction center recorded with the AFM in buffer solution is compared with a surface reconstruction from freeze-dried and unidirectionally, heavy-metal shadowed PSI crystals recorded with the electron microscope. In addition, we demonstrate the feasibility of nanometer scale dissections using the AFM stylus, which gave us the possibility of exploring otherwise hidden surfaces.