The Location of Plastocyanin in Vascular Plant Photosystem I

Abstract

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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“…The low density region will be less deeply embedded in heavy atom stain (being more distant from the grid support film) hence accounting for the lower mean density in this region. Such a differential staining effect has been reported previously (37,38). Comparisons with negatively stained crystals of P-glycoprotein (8, 9) and cryo-electron crystallography of unstained P-glycoprotein crystals 2 suggest that the NBDs are likely to be located in the low density region and may be less well defined than the TMD region.…”

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“…2, panel A). Because the high density region is the side of the molecule in contact with the support film, the differences in the two regions are likely to be because of the well documented 'differential staining' effect (36,37) in which better contrast, and hence higher apparent protein density, is observed in the region closest to the support film. We have previously shown by lectin-gold labeling (27,28) that the surface of the P-gp molecule in contact with the support film corresponds to that exposed at the extracellular face of the membrane.…”

“…Because the NBDs are farther from the grid support film, they are less contrasted by heavy atom stain and less well protected against the damaging effects of the high vacuum and electron beam in the microscope (36,37). Thus, densities in this region are weak, especially for nf-P-gp (Fig.…”

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