Ferredoxin Cross-Links to a 22 kD Subunit of Photosystem I

Abstract

We have used a cross-linking approach to study the interaction of ferredoxin (Fd) with photosystem I (PSI). The cross-linking reagent carbodiimide was found to cross-link spinach Fd to a 22 kilodalton subunit of PSI in both isolated spinach (Spinacia oleracea) PSI complexes and spinach thylakoid membranes.The product had an apparent molecular weight of 38 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified as a cross-linked product using specific antibodies to Fd and the 22 kilodalton subunit. In both a native PSI complex (200 Chl/P700) and a PSI core complex (100 Chl/P700), a second cross-linked product at 36 kilodaltons was seen. The latter cross-reacted with an antibody to Fd but did not cross-react with antibodies directed against the 24.3, 22, 19, 17.3 or 8.5 kilodalton, or psaC subunits of PSI. Its composition remains to be determined. In thylakoids only the 38 kilodalton product was observed along with a cross-linked complex of Fd and Fd:NADP' reductase.Chloroplast thylakoid membranes contain three integral membrane protein complexes which cooperate in the transfer of electrons from water to NADP. These are PSI, PSII, and the Cyt b6-f complex. In this noncyclic electron transport chain, PSI catalyzes light-induced electron transport from reduced plastocyanin to Fd (10).The PSI complex contains P700 as its reaction center Chl and a series of electron acceptors, Ao, A,, Fe-Sx, Fe-SA, and Fe-SB, which accept the electron lost from the reaction center Chl upon photooxidation (10,16 plexes. This cross-linker has previously been used to cross-link Fd to Fd:NADP+ reductase (20) and to cross-link plastocyanin to Cyt f (3). In this study, cross-linked products have been identified by immunoblotting with specific antibodies. Using this approach, a 22 kD subunit ofPSI has been identified as a possible Fd-binding protein in the PSI complex. MATERIALS AND METHODSThylakoid and PSI Preparation. Spinach (Spinacia oleracea) was grown hydroponically in a greenhouse. Washed, deribbed leaves were homogenized in blending buffer (50 mM Tris-HCl [pH 7.8], 0.3 M sucrose, 10 mM NaCl, 5 mM MgCl2). The homogenate was passed through filtering silk and centrifuged for 3 min at 3000 g. The pellets were resuspended in 10 mM MOPS buffer (pH 6.5) for use in cross-linking, or in blending buffer for activity assays. PSI-200 was prepared by the method of Mullet et aL (12). PSI-100 was prepared as described by Ortiz et al. (14). The preparations used in this work contained 185 Chl/P700 and 120 Chl/P700, respectively.Cross-Linking. Ferredoxin was cross-linked to thylakoids as described by Merati and Zanetti (I 1) with the following changes. The cross-linking reaction was carried out in 10 mM MOPS buffer (pH 6.5), 2 mM MgCl2 with 2.5 mm EDC. After quenching the reaction, blending buffer was used to dilute the membranes, which were then pelleted and resuspended in blending buffer.PSI-200 (0.5 mg Chl/mL) was incubated in the presence or absence of 30 jM Fd and 1 mm EDC in the MOPS-MgCl2 buffer f...

Interaction between Photosystem I and Flavodoxin from the Cyanobacterium Synechococcus sp. PCC 7002 as Revealed by Chemical Cross‐Linking

“…In thc casc of fcrrcdoxin, it is generally accepted, although based solely on biochemical evidence, that this acccptor protein is docked exclusively to PsaD during photoreduction (Zanetti and Merati, 1987 ;Zilber and Malkin, 1988;Wynn et al, 1989). The binding of flavodoxin seeins to require the cooperative interaction of several subunits on the cytoplasmic side of PS 1.…”

Modifications to Thylakoid Composition during Development of Maize Leaves at Low Growth Temperatures

“…Antibody reactions were visualized by incubation with a peroxidase-conjugated rabbit antimouse immunoglobulin and development with saturated 4-chloro-I -naphthol solution. Antibodies to a range ofthylakoid proteins were obtained from the following sources: (30); and the aand (-subunits of the coupling factor (58 and 57 kD) from B. Jordan (7). The immunoblots presented for all antigens were produced from gels containing levels of the antigens below those required to saturate the antibodies applied.…”

Purification and membrane topology of PSI‐D and PSI‐E, two subunits of the photosystem I reaction center

“…Background level for each coated immunoglobulin was obtained by revealing with the same immunoglobulin; no Stepwise extraction with NaSCN followed by electrophoresis on a type I gel. After transfer to Immobilon, the membrane was either stained with Coomassie blue (lanes [1][2][3][4] or reacted with a mixture of PSI-D and PSI-E antibodies (lanes [5][6][7][8]. Starting thylakoids (lanes 1, 5 ) ; supcrnatants of successive extractions with 1 M NaSCN (lane 2,6), and 2 M NaSCN (lane 3,7); residual membranes after the final extraction (lanes 4,8).…”