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S8.P24 dicyclihexylcarbodiimide (DCCD)-inhibited ATPase activity of purple Rhodobacter sphaeroides str. MDC6521 (from mineral spring Role of DsrC in the mechanism of sulfite reduction by the waters in Armenian mountains) membrane vesicles for revealing the dissimilatory sulfite reductase DsrAB regulatory pathways of bacterial redox sensing. The membrane + André A. Santosa,SofiaS.Venceslaua,FabianGreinb, Inês Cardoso Pereiraa vesicles showed pronounced H -translocating ATPase activity, aITQB-UNL, Portugal which was inhibited on ~42% by 0.1 mM DCCD. After treatment of – – bUniversity of Bonn, Germany membrane vesicles with 0.5 2 mM DTT an additional increase (~1.6 E-mail: [email protected] 1.8-folds) of DCCD-inhibited ATPase activity was observed. The increase of the ATPase activity by DTT under reducing conditions might be connected with the change of dithiol/disulfide status of this The dissimilatory sulfite reductase (DsrAB) is one of the most enzyme due to the fact that redox-sensitive thiols gain access to the important enzymes in the biogeochemical cycle [1]. It is ATPase. Therefore, the FoF1-ATPase might have an essential role in present in all sulfate reducing microorganisms, in sulfite/thiosulfate/ redox sensing of R. sphaeroides. organosulfonate reducers and in sulfur-oxidizing bacteria. This siroheme-containing enzyme catalyzes the reduction of sulfite, but References despite years of research, the mechanism and physiological products [1] L. Hakobyan, L. Gabrielyan, A. Trchounian, Proton motive force in of sulfite reduction have not been clearly identified. It is not clear Rhodobacter sphaeroides under anaerobic conditions in the dark, why in vitro DsrAB produces a mixture of products including Curr. Microbiol. 62 (2011) 415–419. thiosulfate and trithionate, while the closely-related assimilatory [2] L. Hakobyan, L. Gabrielyan, A. Trchounian, Relationship of proton enzyme reduces sulfite directly to sulfide. More recently, the motive force and the F0F1-ATPase with bio-hydrogen production reduction of sulfite by DsrAB was proposed to involve also the small activity of Rhodobacter sphaeroides: effects of diphenylene iodonium, protein DsrC, which contains two conserved redoxactive cysteines in hydrogenase inhibitor, and its solvent dimethylsulphoxide, J. Bio- a flexible C-terminal arm [2]. The crystal structure of DsrAB in energ. Biomembr. 44 (2012) 495–502. complex with DsrC showed that one of these Cys is located right next to the active site, pointing to the involvement of DsrC in the reduction mechanism [3]. Here, we report recent results from doi:10.1016/j.bbabio.2014.05.106 the investigation on the role of DsrC in the reduction of sulfite by DsrAB, using in vivo and in vitro experiments. Our results show fi that DsrC is directly involved in sul te reduction and permits the S8.P26 identification of the mechanism and physiological product of this important reaction. Study of the respiratory arsenate reductase from halophila definitively clarifies the evolutionary history of this References versatile enzyme [1] Grein F, Ramos AR, Venceslau SS, Pereira IAC (2013) Unifying Barbara Schoepp-Cotheneta, Marielle Bauzanb, Anne-Lise Ducluzeauc, concepts in : Insights from dissimilatory Fabien Pierreld, Wolfgang Nitschkea sulfur metabolism. Biochim Biophys. Acta-Bioenergetics 1827, aBioénergétique et Ingénierie des Protéines, UMR 7281, CNRS, Aix 145–60. Marseille University, IMM FR3479, Marseille, France [2] Venceslau SS, Stockdreher Y, Dahl C, Pereira IAC (2014), The bInstitut de Microbiologie de la Méditerranée, FR3479, F-13402 Marseille “bacterial heterodisulfide” DsrC is a key protein in dissimilatory Cedex 20, France sulfur metabolism, Biochim. Biophys. Acta-Bioenergetics, http:// cBeadle Center, University of Nebraska-Lincoln, 1901 Vine Street, Lincoln, dx.doi.org/10.1016/j.bbabio.2014.03.007. NE 68588-0660, USA [3] Oliveira TF, Vonrheim, Matias PM, Venceslau SS, Pereira IAC, dChimie et Biologie des Métaux, UMR 5249, CEA, CNRS, JF University, Archer M (2008) The crystal structure of a dissimilatory sulfite Grenoble, France reductase bound to DsrC provides novel insights into the E-mail: [email protected] mechanism of sulfate reduction. J. Biol. Chem. 283: 34141.

doi:10.1016/j.bbabio.2014.05.105 The three presently known enzymes responsible for arsenic-using bioenergetic processes are arsenite oxidase (Aio), arsenate reductase (Arr) and alternative arsenite oxidase (Arx), all of which are molybdoenzymes from the vast group referred to as the Mo/W- S8.P25 bisPGD enzyme superfamily. Since arsenite is present in substantial amounts in hydrothermal environments (frequently considered as Dithiothreitol, redox reagent, is affecting proton-translocating vestiges of primordial biochemistry), arsenite-based bioenergetics ATPase activity of Rhodobacter sphaeroides has early on been predicted to be ancient. Conflicting scenarios, a a b Harutyun Sargsyan , Lilit Gabrielyan , Armen Trchounian however, have been put forward proposing either Arr/Arx or Aio as a Department of Microbiology, Microbes and Plants Biotechnology, operating in the ancestral metabolism. Phylogenetic data argue in Faculty of Biology, Yerevan State University, Armenia favour of Aio whereas biochemical and physiological data led several b Department of Microbiology, Plants & Microbes Biotechnology, Faculty authors to propose the Arx/Arr enzyme as the most ancient of Biology, Yerevan State University, Armenia anaerobic arsenite metabolising enzyme. Here we combine phyloge- E-mail: [email protected] netic approaches with physiological and biochemical experiments to demonstrate that the Arx/Arr enzyme could not have been functional The H+-translocating FoF1-ATPase of purple bacteria, the main in the Archaean. We show that Arr reacts with menaquinones to membrane-associated enzyme of bioenergetics relevance, can gen- reduce arsenate whereas Arx reacts with ubiquinone to oxidise erate proton motive force under various conditions [1]. Its role in arsenite, in line with thermodynamic considerations. The phylogeny photo-fermentation and hydrogen production by these bacteria is of the quinone biosynthesis pathway, however, clearly indicates that suggested [2]. In the current work we have studied effects of DL- the ubiquinone pathway is recent. An updated phylogeny of Arr/Arx dithiothreitol (DTT), a redox reagent reducing disulfides, on N,N′- furthermore indicates a recent emergence of this enzyme. We Abstracts e91 therefore conclude that only the metabolism involving Aio could The nature of the carbon metabolism of the extinct primordial have performed anaerobic As redox conversion in the Archaean. organisms is a critical question to understand the origins of life [1,2]. Central to core carbon metabolism is the C1 chemistry involving doi:10.1016/j.bbabio.2014.05.107 folate and its structural analog, methanopterin. Based on the chemical properties of the vents, Lane and Martin [3] put forward a methanogenic origin of archaea, having the Wood–Ljungdahl (WL) pathway as the universal carbon fixation pathway between the two S8.P27 prokaryotic domains. Could it be that an imprint of early chemistry is preserved in the C1 metabolism of modern organisms? We won't Exploring structural/functional relationship in Type II know unless we look, and genomes harbor abundant information. By NADH dehydrogenases studying the distribution and frequency of the enzymes for a a a Filipa V. Sena , Ana P. Batista , Bruno C. Marreiros , methanopterin and folate biosynthesis within sequenced genomes b c a Margarida Bastos , Eurico J. Cabrita , Manuela M. Pereira [4,5], we found that these distinct biosynthetic routes are unrelated a Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, across the two domains, indicating that the corresponding pathways Av. Da República EAN, 2780-157 Oeiras, Portugal arose independently. This dichotomy is also observed in the b CIQ (UP) Departmento de Química, Faculdade de Ciências, Universidade structurally unrelated enzymes and different organic cofactors that do Porto, P-4169-007 Porto, Portugal methanogens (archaea) and acetogens (bacteria) use to perform c REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, methyl synthesis in their H4F- and H4MPT-dependent versions, Universidade Nova de Lisboa, Caparica, Portugal respectively, of the WL pathway. The data suggests that, in contrast E-mail: [email protected] to the ancestry of the acetyl synthesis segment, the methyl segment of the WL pathway evolved in a later stage, after the divergence of Type-II NADH:quinone oxidoreductases (NDH-II) are membrane bacteria and archaea, which independently invented genetically- proteins involved in respiratory chains. NDH-II performs the same encoded means to synthesize methyl groups via enzymatic reactions. reaction as Complex I, but does not contribute to the generation of the ion electrochemical potential. Both enzymes may be expressed References by the same organism according to its metabolic demands. Some [1] Y. Liu, L.L. Beer, W.B. Whitman, Methanogens: a window into pathogenic bacteria contain only genes encoding NDH-II and in ancient sulfur metabolism. Trends Microbiol. 20 (2012) 251–258 animal mitochondria only Complex I is expressed. The study of NDH- [2] G. Fuchs, Alternative pathways of carbon dioxide fixation: II gained a new enthusiasm after the publication of two yeast insights into the early evolution of life? Annu Rev Microbiol. 65 structures, which brought additional discussions also due to their (2011) 631–658. apparently contradictory data, concerning the quinone binding site [3] N. Lane, W.F. Martin, The origin of membrane bioenergetics. Cell. [1–2]. The first crystal structure of a bacterial NDH-II enzyme was 151 (2012) 1406–1416. reported this year [3] and revealed unique binding sites for the [4] V. de Crécy-Lagard, B. El Yacoubi, R. Díaz de la Garza, A. Noiriel, substrates. Still the interaction of NDH-II with the substrates, A.D. Hanson, Comparative genomics of bacterial and plant folate including the localization of the binding-sites remains unclear. synthesis and salvage: predictions and validations. BMC Geno- This work aims to study protein–substrate interaction of NDH-II with mics. 8 (2012) 245. different quinones, in order to elucidate the structure/function relation, [5] F.L. Sousa, W.F. Martin, Biochemical fossils of the ancient namely the determinants for specificity for different substrates. A transition from geoenergetics to bioenergetics in prokaryotic comparative study, through a range of methodologies, of the differences one carbon compound metabolism. BBA — Bioenergetics. (2014) between ubiquinone and menaquinone bindings was performed. in press. Implications on the catalytic mechanism of NDH-II are discussed. doi:10.1016/j.bbabio.2014.05.109 References [1] M. Iwata, Y. Lee, T. Yamashita, T. Yagi, S. Iwata, A.D. Cameron, M.J. Maher, The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble S8.P29 substrates, Proc Natl Acad Sci U S A 109 (2012) 15247–15252. [2] Y. Feng, W. Li, J. Li, J. Wang, J. Ge, D. Xu, Y. Liu, K. Wu, Q. Zeng, J.W. Direct observation of CWD bacteria reproduction Wu, C. Tian, B. Zhou, M. Yang, Structural insight into the type-II Kazuhito Tabata, Takao Sogo, Hiroyuki Noji mitochondrial NADH dehydrogenases, Nature 491 (2012) 478–482. The University of Tokyo, Japan [3] A. Heikal, Y. Nakatani, E. Dunn, M.R. Weimar, C.L. Day, E.N. Baker, J.S. E-mail: [email protected] Lott, L.A. Sazanov, G.M. Cook, Structure of the bacterial type II NADH dehydrogenase: a monotopic membrane protein with an essential Cell wall defective (CWD) bacteria are made by using lysozymes role in energy generation, Mol Microbiol 91 (2014) 950–964. to disrupt the cell wall and are insensitive to β lactam antibiotics [1]. Understanding the mechanism of this resistance should provide fi doi:10.1016/j.bbabio.2014.05.108 signi cant insight on how CWD bacteria grow, reproduce and proliferate [2–4]. Accordingly, we observed different properties of CWD bacteria, such as growth, metabolism, and protein synthesis. We prepared CWD Escherichia coli and cultured them in ampicillin. S8.P28 The CWD E. coli did not divide but grew, reaching a maximum diameter of 10 μm at 8 h of culturing. Consistent with this Independent origins of the methyl segments of the observation, protein synthesis and metabolic activity were observed Wood–Ljungdahl pathway for 8 h. Upon removing the ampicillin from the culture, the CWD Filipa L. Sousa, William F. Martin E. coli began to deform and divide. Furthermore, the divided CWD Institute of Molecular Evolution, Germany E. coli was only 3–5 μm diameters, suggesting the division mecha- E-mail: fi[email protected] nism did not function beyond this size. Finally, we will also discuss