[7] J.G. Okun, P. Lummen, U. Brandt, J. Biol. Chem. 274 (1999) 2625–2630. doi:10.1016/j.bbabio.2010.04.082 1P.35 The structure of complex I from the hyperthermophilic eubacterium Aquifex aeolicus Guohong Peng 1 , Ulrich Ermler 1 , Todd Clason 2 , Sandra Bornemann 3 , Tanja Hedderich 1 , Teresa Ruiz 2 , Bjoern Meyer 3 , Michael Radermacher 2 , Michael Karas 3 , Hartmut Michel 1 1 Max Planck Institute of Biophysics, Department of Molecular Membrane Biology, Frankfurt am Main 60438, Germany 2 University of Vermont, College of Medicine, Department of Molecular Physiology and Biophysics, Burlington, VT 5405, USA 3 Chemical and Pharmaceutical Sciences, Institute for Pharmaceutical Chemistry, J.W. Goethe University of Frankfurt. Frankfurt 60439, Germany E-mail: Hartmut.Michel@biophys.mpg.de (H. Michel), Guohong.Peng@biophys.mpg.de (G. Peng) Complex I from Aquifex aeolicus is highly stable and active. Image analysis and 2D and 3D reconstruction by electron micrographs revealed a complete complex I particle of typical L-shape, and a pronounced invariant angle (90°) between the cytoplasmic arm [1–2] and the membrane arm. It showed many details in its external arm. The isoforms of the complex have been detected by mass spectrometry. So far, the subunits in the hydrophilic domain could be clearly assigned to two isoforms. The partial structure of one isoform of Aquifex complex I containing all subunits of hydrophilic domain has been determined by X-rayat a 2.9 Å resolution. Interestingly, Aquifex complex I contains one extra iron sulfur cluster, which is not found in that of E. coli and T. thermophilus. These data allow us to describe and discuss the mechanistic hypotheses and models of bacterium complex I [3–5]. References [1] G.H. Peng, G. Fritzsch, V. Zickermann, H. Schägger, R. Mentele, F. Lottspeich, M. Bostina, M. Radermacher, R. Huber, K.O. Stetter, H. Michel, Biochemistry 42 (2003) 3032–3039. [2] T. Clason, T. Ruiz, H. Schägger, G. Peng, V. Zickermann, U. Brandt, H. Michel, Radermacher, J. Struct. Biol.169 (2010) 81–88. [3] L.A. Sazanov, P. Hinchliffe, Science 311 (2006) 1430–1436. [4] J.M. Berrisford, L.A. Sazanov, J. Biol. Chem. 284 (2009) 29773–29783. [5] J. Hirst, Biochem. J. 425 (2010) 327–339. doi:10.1016/j.bbabio.2010.04.083 1P.36 A systematic approach to membrane-protein reconstitution in liposomes, applied to the M2 protein of Influenza virus A Thom Leiding 1 , Jonas Martinsson 1 , Sergei Vinogradov 2 , Cecilia Hägerhäll 1 , Sindra Peterson Årsköld 1 1 Center for Molecular Protein Science, Lund University, Sweden 2 University of Pennsylvania, USA E-mail: sindra.peterson_arskold@biochemistry.lu.se We present an improved methodology for production of large unilamellar vesicles and reconstitution of membrane-proteins, using gradual detergent removal. We also present two novel membrane- impermeable pH sensors, the porphyrin-based Glu3 and TCHP (Leiding et al., 2009, Anal. Biochem. 388: 296–305). The solubiliza- tion behavior of vesicles in different detergents is reported, and the effect of protein-to-lipid concentration on passive ion permeability of the liposomes. The effects of cholesterol and lipid composition on vesicle integrity are also explored — all for the purpose of under- standing and optimizing the protein reconstitution process. As a proof of concept, successful unidirectional reconstitution of the Influenza protein A/M2 is reported. The integrity of the proteolipo- somes allowed detailed, quantitative data collection over tens of minutes, providing a wealth of new information on ion flux through the protein (cf. Thom Leiding's poster). This reliable reconstitution method, together with pH sensors that stay within vesicles and a semi-automated titration and data-analysis system, provides a strong platform for investigating proton-translocating bioenergetic complexes. doi:10.1016/j.bbabio.2010.04.084 1P.37 A novel c-type cytochrome transfers electrons between sulfite oxidase and cytochrome c 552 in the respiratory chain of Thermus thermophilus Sylvain Robin 1 , Marzia Arese 2 , Elena Forte 2 , Paolo Sarti 2 , Alessandro Giuffré 2 , Tewfik Soulimane 1 1 Chemical and Environmental Science Department, Materials and Surface Science Institute, University of Limerick, Ireland 2 Department of Biochemical Sciences and CNR Institute of Molecular Biology and Pathology, Sapienza University of Rome, Italy E-mail: tewfik.soulimane@ul.ie We here describe a novel c-type cytochrome from the extreme thermophile Thermus thermophilus. N-terminal sequencing of the purified protein led to the identification of the corresponding gene TTHA1326. The 23 kDa cytochrome possesses two heme c binding sites and demonstrates a high sequence identity to cytochrome c 552 , the substrate of the ba 3 -type cytochrome c oxidase. Because of the low yield, we have succeeded in its recombinant production in E. coli with the simultaneous expression of the ccm genes involved in the maturation of cytochrome c in the same organism. We have generated milligram quantities of the holo-protein allowing the investigation of its properties and physiological function. There is no evidence that cytochrome c 550 acts as an electron shuttle between the bc complex and Thermus cytochrome c oxidases. We have shown that, surprisingly, cytochrome c 550 clearly mediates electrons to cytochrome c 552 . Further analysis of the putative operon encoding the protein led to the identification of a potential electron donor namely sulfite oxidase. In order to assess the subsequent electron transfer, sulfite oxidase (SO) TTHA1325 was produced recombinantly in E. coli and was shown to utilize the cytochrome c 550 as the electron acceptor following oxidation of sulfite. To the best of our knowledge, this is the first characterization of the sulfite respiration system from a thermophilic bacterium. doi:10.1016/j.bbabio.2010.04.085 1P.38 Functional analysis of respiratory complex I (NADH:ubiquinone oxidoreductase) in the early-branching eukaryote Trypanosoma brucei Achim Schnaufer 1 , Meredith Heestand 2 , Brian Panicucci 2 , Sachin Surve 2 , Marilyn Parsons 2,3 1 Institute of Immunology & Infection Research, University of Edinburgh, UK 2 Seattle Biomedical Research Institute, USA 3 Department of Global Health, University of Washington, USA E-mail: achim.schnaufer@ed.ac.uk The protozoan parasite Trypanosoma brucei alternates between a mammalian host and an insect vector, and these environmental changes have resulted in dramatic regulation of the organism's Abstracts 22