Discovery of Native Metal Ion Sites Located on the Ferredoxin Docking Side of Photosystem I † Lisa M. Utschig,* Lin X. Chen, and Oleg G. Poluektov Chemical Sciences and Engineering DiVision, Argonne National Laboratory, Argonne, Illinois 60439 ReceiVed January 8, 2008 ABSTRACT: Photosystem I (PSI) is a large membrane protein that catalyzes light-driven electron transfer across the thylakoid membrane from plastocyanin located in the lumen to ferredoxin in the stroma. Metal analysis reveals that PSI isolated from the cyanobacterial membranes of Synechococcus leopoliensishas a near-stoichiometric 1 molar equiv of Zn 2+ per PSI monomer and two additional surface metal ion sites that favor Cu 2+ binding. Two-dimensional hyperfine sublevel correlation (HYSCORE) spectroscopy reveals coupling to the so-called remote nitrogen of a single histidine coordinated to one of the Cu 2+ centers. EPR and X-ray absorption fine structure (XAFS) studies of 2Cu-PSI complexes reveal the direct interaction of ferredoxin with the Cu 2+ centers on PSI, establishing the location of native metal sites on the ferredoxin docking side of PSI. On the basis of these spectroscopic results and previously reported site-directed mutagenesis studies, inspection of the PSI crystal structure reveals a cluster of three highly conserved residues, His(D95), Glu(D103), and Asp(C23), as a likely Cu 2+ binding site. The discovery of surface metal sites on the acceptor side of PSI provides a unique opportunity to probe the stromal region of PSI and the interactions of PSI with its reaction partner, the soluble electron carrier protein ferredoxin. Photosynthetic reaction center proteins (RCs) 1 are finely tuned molecular systems optimized for solar energy conver- sion. In RCs, photoinduced rapid, sequential electron transfer reactions result in the formation of a charge-separated state that is linked to important secondary reaction sequences, such as proton transfer or electron transfer with mobile charge carriers. Previously, we discovered a surface metal ion site on the purple photosynthetic bacterial RC (bRC) that modulates proton-coupled electron transfer (1, 2). In bRCs, light-induced electron transfer terminates in the electron transfer between two quinone molecules, Q A and Q B , and this electron transfer reaction is coupled to proton uptake. Zn 2+ binds near the protein surface to a site located ∼18 Å below the Q B binding pocket (3) and slows interquinone electron transfer (1) and proton uptake by Q B (4). Subse- quently, proton gateways in the bRC (5) and other proteins have been identified via surface Zn 2+ sites (6). The impor- tance of surface Zn 2+ protein sites has led us to examine the possibility that such metal ion sites exist in the photosystem I (PSI) RC. In oxygenic photosynthesis of higher plants, cyanobacteria, and algae, PSI catalyzes light-driven electron transfer across the thylakoid membrane from plastocyanin located in the lumen to ferredoxin in the stroma (7, 8). PSI is a large, membrane protein complex composed of 12 protein subunits and 127 cofactors. In PSI, photoexcitation of the primary electron donor, P700 (a dimer of cholorophyll molecules), initiates electron transfer to two spectroscopically identified electron acceptors, A 0 , a chlorophyll molecule, and A 1 ,a phylloquinone. From A 1 - , the electron is transferred to the [4Fe-4S] cluster F X , and further to F A and F B , two [4Fe-4S] clusters held within the extrinsic protein subunit PsaC. The electron is then transferred to ferredoxin, a small [2Fe-2S] protein that shuttles the reducing equivalents from PSI to several metabolic pathways. Three protein subunits, PsaC, PsaD, and PsaE, form the stromal hump of PSI and are involved in the docking of ferredoxin (7–9). To date, the only reported transition metal ion in PSI is the Fe found in the clusters. Herein, we report the discovery of native surface metal ion sites on the acceptor side of the PSI RC and provide spectroscopic evidence of the interaction of these sites with the soluble electron carrier protein ferredoxin. EXPERIMENTAL PROCEDURES Purification of Photosystem I. Cells of Synechococcus leopoliensis (UTEX625) were grown in Ac medium (10) at ∼40 °C. Thylakoid membranes and PSI complexes were prepared according to the procedures of Rögner et al. (11). PSI was purified through the sucrose gradient step; additional chromatography steps that might strip native bound metal ions from PSI were eliminated. The lower dark green band from the sucrose gradient was dialyzed overnight into 20 mM Hepes (pH 7.9), 25 mM MgSO 4 , and 0.03% -DM (n- dodecyl -D-maltopyranoside, Anatrace) and concentrated with Centriprep-50 (Amicon) devices. PSI purified with Tris/ † This work was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences, under Contract DE-AC02–06CH11357. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357. * To whom correspondence should be addressed. Phone: (630) 252- 3544. Fax: (630) 252-9289. E-mail: utschig@anl.gov. 1 Abbreviations: RC, reaction center; bRC, bacterial reaction center; PSI, photosystem I; HYSCORE, two-dimensional hyperfine sublevel correlation; EPR, electron paramagnetic resonance; XAFS, X-ray absorption fine structure; cw, continuous wave; ESE, electron spin–echo. Biochemistry 2008, 47, 3671–3676 3671 10.1021/bi800038d CCC: $40.75  2008 American Chemical Society Published on Web 02/23/2008