Asymmetric Dimeric Structure of Ferredoxin-NAD(P) + Oxidoreductase from the Green Sulfur Bacterium Chlorobaculum tepidum: Implications for Binding Ferredoxin and NADP + Norifumi Muraki 1,2 †, Daisuke Seo 3 ⁎†, Tomoo Shiba 1 , Takeshi Sakurai 3 and Genji Kurisu 2,4 ⁎ 1 Department of Life Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan 2 Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan 3 Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan 4 Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan Received 26 December 2009; received in revised form 9 June 2010; accepted 11 June 2010 Available online 18 June 2010 Ferredoxin-NAD(P) + oxidoreductase (FNR) catalyzes the reduction of NAD(P) + to NAD(P)H with the reduced ferredoxin (Fd) during the final step of the photosynthetic electron transport chain. FNR from the green sulfur bacterium Chlorobaculum tepidum is functionally analogous to plant- type FNR but shares a structural homology to NADPH-dependent thioredoxin reductase (TrxR). Here, we report the crystal structure of C. tepidum FNR to 2.4 Å resolution, which reveals a unique structure– function relationship. C. tepidum FNR consists of two functional domains for binding FAD and NAD(P)H that form a homodimer in which the domains are arranged asymmetrically. One NAD(P)H domain is present as the open form, the other with the equivalent NAD(P)H domain as the relatively closed form. We used site-directed mutagenesis on the hinge region connecting the two domains in order to investigate the importance of the flexible hinge. The asymmetry of the NAD(P)H domain and the comparison with TrxR suggested that the hinge motion might be involved in pyridine nucleotide binding and binding of Fd. Surprisingly, the crystal structure revealed an additional C-terminal sub-domain that tethers one protomer and interacts with the other protomer by π-π stacking of Phe337 and the isoalloxazine ring of FAD. The position of this stacking Phe337 is almost identical with both of the conserved C-terminal Tyr residues of plant-type FNR and the active site dithiol of TrxR, implying a unique structural basis for enzymatic reaction of C. tepidum FNR. © 2010 Elsevier Ltd. All rights reserved. Edited by M. Guss Keywords: ferredoxin-NAD(P) + reductase; photosynthesis; electron transfer complex; X-ray crystallography; thioredoxin reductase Introduction Ferredoxin-NAD(P) + reductase (FNR, EC 1.18.1.2) is a ubiquitous ferredoxin (Fd)-dependent enzyme containing flavin adenine dinucleotide (FAD) as a prosthetic group, which generally catalyzes the reversible redox reaction between Fd and NAD(P) + / NAD(P)H. On the basis of phylogenic and structural information, FNRs are classified into two distinct families; plant-type and glutathione reductase (GR)- type. 1 FNR, which is representative of the plant type, is a plastidic-type localized in chloroplast 2–4 or non- *Corresponding authors. E-mail addresses: dseo@cacheibm.s.kanazawa-u.ac.jp; gkurisu@protein.osaka-u.ac.jp. † N.M. and D.S. contributed equally to this work. Present address: T. Shiba, Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Abbreviations used: FNR, ferredoxin-NAD(P) + reductase; Fd, ferredoxin; GR, glutathione reductase; AdR, adrenodoxin reductase; ONFR, oxygenase-coupled NADH-ferredoxin reductase; RC, photoreaction center; TrxR, thioredoxin reductase; FO, flavin-oxidizing; FR, flavin-reducing; WT, wild type. doi:10.1016/j.jmb.2010.06.024 J. Mol. Biol. (2010) 401, 403–414 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.