Structure of Synechococcus elongatus [Fe 2 S 2 ] Ferredoxin in Solution ² Bettina Baumann, ‡,§ Heinrich Sticht, ‡,| Manuela Scha ¨rpf, ‡ Martin Sutter, ⊥ Wolfgang Haehnel, ⊥ and Paul Ro ¨sch* ,‡ Lehrstuhl fu ¨ r Biopolymere, UniVersita ¨ t Bayreuth, UniVersita ¨ tsstrasse 30, D-95447 Bayreuth, Germany, and Lehrstuhl fu ¨ r Biochemie der Pflanzen, Institut fu ¨ r Biologie II, Albert-Ludwigs-UniVersita ¨ t, Scha ¨ nzlestrasse 1, D-79104 Freiburg, Germany ReceiVed May 15, 1996; ReVised Manuscript ReceiVed July 8, 1996 X ABSTRACT: Ferredoxins of the [Fe 2 S 2 ] type function in photosynthetic electron transport as essential electron acceptors of photosystem I. The solution structure of the 97 amino acid ferredoxin from the thermophilic cyanobacterium Synechococcus elongatus was determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. The structure consists of a four-stranded parallel/ antiparallel -sheet, a short two-stranded antiparallel -sheet, and three short helices. The overall structure is similar to the structure of the ferredoxin from Anabaena. In contrast to related ferredoxins from mesophilic organisms, this thermostable protein contains a salt bridge inside a 17-amino acid hydrophobic core. Ferredoxin (Fd) 1 is an essential electron carrier transferring electrons from the membrane bound complex of photosystem I to the Fd:NADP + oxidoreductase (EC 1.18.1.2) which, in turn, reduces NADP + for CO 2 fixation in photosynthesis. Ferredoxin I in oxygenic, photosynthetic organisms is a soluble acidic protein with a [Fe 2 S 2 ] center that transfers one electron, while in anoxygenic procaryotic organisms the major Fd is a [Fe 4 S 4 ] protein (Knaff & Hirasawa, 1991). Cells with a deletion of the petFI gene encoding Fd I were not viable (van den Plas et al., 1988) showing that its function is indispensable. Fd is reduced by one of the two [Fe 4 S 4 ] centers of the PsaC subunit at the stromal side of the photosystem I complex. This electron transfer is only possible on simultaneous contact with the positively charged PsaD subunit (Zanetti & Merati, 1987). The role of the peripheral PsaE subunit of photosystem I may be the stabilization of the electron transfer to Fd (Weber & Strotmann, 1993; Rousseau et al., 1993) or facilitation of the cyclic electron transfer (Yu et al., 1993). Fd I appears to serve a central role in shuttling the reducing equivalents originating from H 2 O not only to the Fd:NADP + oxi- doreductase, but also to other enzymes such as the fdx:nitrite oxidoreductase (EC 1.7.7.1), glutamate synthase (EC 1.4.7.1), sulfate reductase, or the Fd:thioredoxin reductase. These multiple interactions suggest a docking site of Fd at these enzymes, possibly similar to a mirror-image structure of Fd contributing negatively charged groups for an elec- trostatically stabilized complex formation (Knaff & Hirasawa, 1991) while small differences may control the electron transfer channeled to the different pathways. Recently the structure of photosystem I from Synechococcus elongatus has been solved at 6 Å resolution (Krauss et al., 1993). Improvement of the structure may reveal details of the binding site of Fd (Fromme and Saenger, personal com- munication). Therefore, the structure of Fd from this organism is of particular interest. In general, the known [Fe 2 S 2 ] ferredoxins from plants, algae, and cyanobacteria consist of 93-99 amino acid residues (Matsubara & Saeki, 1992) and are highly homolo- gous among each other (Table 1). The iron-sulfur clusters are attached to the protein chain via cystein residues. For example, in the 97-amino acid S. elongatus ferredoxin (Fd Se ) the [Fe 2 S 2 ] cluster active in the electron transfer is attached to the protein chain via the sulfhydryl groups of Cys40, Cys45, Cys48, and Cys78 (Hase et al., 1983). Particularly the cluster surroundings are well conserved in the various ferredoxins (Fukuyama et al., 1980). This high sequence homology is even more remarkable considering the fact that the cyanobacteria were among the first photosynthetic organisms and thus have been around for more than three billion years (Meyer, 1988). The crystal structure of four ferredoxins of the [Fe 2 S 2 ] type from different organisms is known: Fukuyama et al. published the first crystal structure of Fd from Spirulina platensis (Fukuyama et al., 1980). Similar crystal structures have been found for Aphanothece sacrum (Tsukihara et al., 1990), Anabaena PCC 7120 (Rypniewski et al., 1991), and Equisetum arVense (Ikemizu et al., 1994). The nuclear magnetic resonance structure of the [Fe 2 S 2 ] Fd from Syn- echocystis sp. PCC 6803 (Lelong et al., 1995) and of putidaredoxin from Pseudomonas putida (Pochapsky et al., 1994), which is only distantly related to the cyanobacterial ferredoxins, were recently determined. In addition, the 1 H, ² Support by Deutsche Forschungsgemeinschaft SFB 388/A1 is gratefully acknowledged. The atomic coordinates (code 1ROE) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY. * To whom correspondence should be adressed: Tel: ++49 921 553540. Fax: ++49 921 553544. E-mail: paul.roesch@uni-bayreuth. de. ‡ Universita ¨t Bayreuth. § Present address: Institut fu ¨r Organische Chemie, Johann-Wolfgang- Goethe-Universita ¨t Frankfurt, Marie-Curiestrasse 11, D-60439 Frank- furt, Germany. | Present address: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England. ⊥ Albert-Ludwigs-Universita ¨t. X Abstract published in AdVance ACS Abstracts, September 1, 1996. 1 Abbreviations: 2D, two dimensional; clean-TOCSY, TOCSY with suppression of NOESY-type crosspeaks; COSY, correlated spectros- copy; DQF-COSY, double quantum filtered COSY; DSS, 2,2-dimethyl- 2-silapentane-5-sulfonic acid; DSSP, definition of secondary structure of proteins; Fd, ferredoxin; FdSe, Synechococcus elengatus [Fe2S2] ferredoxin; MD, molecular dynamics; NMR, nuclear magnetic reso- nance; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; ppm, parts per million; RMSD, root mean square deviation; SA, simulated annealing; TOCSY, total correlation spectroscopy; TPPI, time proportional phase incrementation. 12831 Biochemistry 1996, 35, 12831-12841 S0006-2960(96)01144-0 CCC: $12.00 © 1996 American Chemical Society