Short crystallization paper Crystallisation and preliminary structure determination of a NADH:quinone oxidoreductase from the extremophile Acidianus ambivalens Jose ´ A. Brito a , Tiago M. Bandeiras a , Miguel Teixeira a , Clemens Vonrhein b , Margarida Archer a, * a Instituto de Tecnologia Quı ´mica e Biolo ´ gica, Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal b Global Phasing Ltd., Sheraton House, Castle Park, Cambridge CB 3 OAX, England Received 21 July 2005; received in revised form 8 September 2005; accepted 27 September 2005 Available online 24 October 2005 Abstract NADH:quinone oxidoreductases (NDHs), constitute one of the electron entry points into membrane-bound respiratory chains, oxidising NADH and reducing quinones. Type-II NDHs (NDH-2) are functionally unable to translocate protons and are typically constituted by a single ¨50 kDa subunit lacking iron – sulfur clusters and containing one flavin as the sole redox centre. No three dimensional crystal structure is yet available for NDHs. We describe the crystallisation and preliminary structure determination of a NDH-2 that contains a covalently bound FAD, isolated from the membrane fraction of Acidianus ambivalens , a hyperthermoacidophilic archaeon capable of growing at 80 -C and pH 2.0. NDH- 2 was solubilised with the detergent n-dodecyl-h-d-maltoside and crystallised using ammonium phosphate as precipitant. The structure was solved by MIRAS using Pt and I derivatives. D 2005 Elsevier B.V. All rights reserved. Keywords: Crystallisation; NADH:quinone oxidoreductase; Membrane protein; Aerobic respiratory chain; Extremophile; Archaea Mitochondrial respiratory chains are classically composed of four membrane-associated enzymatic complexes: NADH: quinone oxidoreductase (NDH); succinate:quinone oxidore- ductase (SQR); quinol:cytochrome c oxidoreductase and cyto- chrome c :oxygen oxidoreductase. Apart from SQR, all complexes couple electron transfer with ion translocation generating a potential difference across the membrane which is used by the ATP synthase to produce ATP and ADP, and to drive the movement of substrate molecules against a concen- tration gradient. The NADH:quinone oxidoreductase (NDH) family can be divided into three types of enzymes: complex I; type-2 NDH and Na + -NDH [1–4]. In contrast with the other components of the aerobic respiratory chain, no X-ray structure is yet known for NADH:quinone oxidoreductases by NDHs, the electron entry point in aerobic respiratory chains. Type-2 NADH:quinone oxidoreductases (NDH-2) are 50 kDa monomers, containing no iron – sulfur clusters and one flavin molecule as the sole redox cofactor [1]. These enzymes do not translocate protons and according to the presence of one or two dinucleotide-binding motifs [5] and one EF-hand motif (calcium binding site) in their primary structures, these enzymes have been divided in three families: A, B and C [6]. These enzymes play a key role in bioenergetics, since many organisms rely solely on them to oxidize NADH and reduce the quinone pool. Moreover, they have recently been proposed to functionally substitute complex I-deficient mito- chondria, which makes NDHs putative targets for gene therapy [7]. The components of the respiratory chain of Acidianus ambivalens , an hyperthermoacidophilic archaeon (T opt ¨80 -C, pH opt ¨2) have been extensively characterised [8–11]. The organism apparently does not contain a complex I-like enzyme but instead has a membrane-bound type C NDH-2, an enzyme of 47 kDa, containing a covalently bound flavin with a reduction potential of ¨70 mV [8,12]. Cells of Acidianus ambivalens (DSM 3772) were grown aerobically in 10 L glass flasks by batch culture, at 80 -C and pH 2.5 as described in [13]. The growth media was completed with the addition of 0.02% of yeast extract. Growth was followed by monitoring the absorbance band at 546 nm after removal of the suspended sulfur by filtration. The pH was also measured in order to follow the sulphuric acid production. 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.09.015 * Corresponding author. E-mail address: archer@itqb.unl.pt (M. Archer). Biochimica et Biophysica Acta 1764 (2006) 842 – 845 http://www.elsevier.com/locate/bba